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Keywords:

  • Apoptosis-inducing factor;
  • IL-4;
  • Mast cells;
  • STAT6

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

IL-4 can suppress mast cell development from mouse spleen, bone marrow and peritoneal cells by an indirect process that is dependent on the presence of macrophages. Mast cells undergo apoptosis when exposed to supernatants collected from cultures of IL-4-stimulated peritoneal cells due to the IL-4-induced production of an apoptosis-inducing factor in the cultures. This effect of IL-4 is shown to be dependent on STAT6 signaling, because IL-4 and IL-13 do not suppress mast cell development from the spleen and peritoneal cells of STAT6–/– mice. Moreover, supernatants from cultures of IL-4- and IL-13-stimulated peritoneal cells of STAT6–/– mice do not exhibit apoptosis-inducing activity. We confirm, by using deficient mice, neutralizing antibodies and recombinant cytokines, that IL-4-induced apoptosis is not related to the well-known apoptosis-inducing factors Fas, Fas ligand, TNF-α, TRAIL, TGF-β or perforin. These results demonstrate a novel mechanism whereby IL-4 and IL-13 can suppress mast cell development by inducing the production of an apoptosis-inducing factor from macrophages.

Abbreviations:
LIF:

leukemia inhibitory factor

OSM:

oncostatin M

PKO:

perforin knock-out

PS:

phospholipid phosphatidylserine

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Mast cells are derived from precursors that are present in very small numbers in bone marrow and blood as well as spleen 13. Under normal conditions, these precursors give rise to mature mast cells, which appear to be long-lived cells that are maintained at relatively constant numbers in various tissues. Mast cell growth factors, such as IL-3, IL-4, IL-9, IL-10 and SCF 411, play critical roles in maintaining numbers of mast cells, because withdrawal of these growth factors, especially SCF and IL-3, can result in mast cell apoptosis 12, 13. The immune system relies on cell death to maintain homeostasis and avoid disease. While apoptosis induced by various apoptotic factors plays a central role in shaping the repertoire of mature cells of the immune system, little is known about apoptosis-inducing factors that down-regulate mast cells 14, 15.

IL-4 is a multifunctional Th2 cell-associated cytokine that, through STAT6-dependent signaling, plays a critical role in the regulation of immune responses 1618. In addition to promoting Ig class switching from IgM to IgE in B cells 19, under certain circumstances IL-4 can promote mast cell proliferation and differentiation by synergizing with IL-3 and/or SCF 5, 6, 15, 20.

We unexpectedly found that the numbers of mast cells that develop from mouse spleen cells are inversely proportional to IL-4 levels in the cultures 21. Moreover, others have reported that IL-4 can directly induce apoptosis in both mouse 22, 23 and human 24 mast cells, at least under certain conditions in vitro. Taken together, these results reveal that IL-4 is able to function as an inhibitor of mast cell development. Nevertheless, the precise mechanisms by which IL-4 can suppress mast cell development are not fully understood. In this study, we provide evidence that IL-4 can suppress mast cell development from bone marrow and peritoneal cells, as well as from spleen cells, and that IL-4 exerts this effect by inducing STAT6-dependent production of an unknown apoptosis-inducing factor.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Suppression of mast cell development from mouse peritoneal and bone marrow cells by IL-4

We previously reported that IL-4 is a potent inhibitor of mast cell development from mouse spleen cells 21. To rule out the possibility that IL-4 specifically inhibits mast cell development from spleen cells, we tested the effect of IL-4 on mast cell development from peritoneal and bone marrow cells. In the presence of IL-3 and SCF, large numbers of mast cells developed from such cells during 8–12 days of culture. Furthermore, this mast cell development was suppressed by IL-4 in a dose- and time-dependent manner (Fig. 1). These results confirm that suppression of mast cell development by IL-4 is a general phenomenon and is not specific to mast cell development from spleen cells.

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Figure 1. Dose- and time-dependent suppression of mast cell development from peritoneal or bone marrow cells by IL-4. Peritoneal cells (105/mL, black bars) or bone marrow cells (5 × 105/mL, hatched bars) from C57BL/6 mice were cultured with 10 ng/mL rIL-3 and 50 ng/mL rSCF, and rIL-4 was added at various concentrations (A) or at a concentration of 10 ng/mL on day 0, 2, 4 or 6 (B). Mast cell numbers were counted on day 12 (*p<0.05 compared to the corresponding value for cells of the same group cultured without rIL-4).

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IL-4 can enhance mast cell development through effects on mast cell precursors as well as on purified mast cells

One possible explanation for the time-dependent suppression of mast cell development is that IL-4 may act at various stages of their differentiation and down-regulate their development by affecting mast cell precursors. To evaluate this theory, c-kit+ cells (including mast cell precursors) were separated from spleen cells by MACS and seeded in a semi-solid culture to observe mast cell development. In the presence of IL-3 and SCF, mast cell development from the c-kit+ cells was promoted by IL-4. IL-4, again in the presence of IL-3 and SCF, also enhanced the growth of purified mast cells and a cell line representing immature mast cells (IC-2 cells). Thus, in agreement with previous reports, IL-4 was able to promote the growth of mast cells and their precursors (Table 1).

Table 1. Effects of IL-4 on numbers of c-kit+ cells, IC-2 cells and purified mast cells
Cell type and cell number per cultureCytokinesa)Viable cells/mast cells per culture (× 104)b)Promotion(%)
  1. a) Cytokines: 20 ng/mL rIL-3, 50 ng/mL rSCF and 10 ng/mL rIL-4

  2. b) The c-kit+ cells were cultured in semi-solid medium, and mast cells were counted on day 14. IC-2 cells and purified mast cells were cultured in liquid medium, and viable cells were counted on day 3.

  3. c)p<0.05 vs. corresponding value for cells of the same type cultured without rIL-4

c-kit+ cells, 104rIL-3 + rSCF31.1±3.7
rIL-3 + rSCF + rIL-466.6±4.5c)114
Purified mast cells, 5 × 104rIL-3 + rSCF80.5±10.8
rIL-3 + rSCF + rIL-4104.6±3.6c)30
IC-2 cells, 2.5 × 104rIL-3 + rSCF21.7±4.6
rIL-3 + rSCF + rIL-448.1±0.6c)122

Effect of macrophages on IL-4-induced suppression of mast cell development

To evaluate whether the suppressive effect of IL-4 is an indirect process, different densities of peritoneal cells were cultured in semi-solid medium with IL-3 and SCF in the presence of IL-4. IL-4 promoted the development of mast cells from peritoneal cells at a density of 104 cells per culture but completely suppressed mast cell development when the peritoneal cells were incubated at 5 × 104 cells per culture (Table 2).

Table 2. Effects of cell number on IL-4-induced suppression of mast cell development from peritoneal cells
Number of peritoneal cellsCytokinesMast cells/culturea) (× 104)Promotion or suppression (%)
  1. a) Various numbers of peritoneal cells from normal C57BL/6 mice were cultured in a semi-solid medium containing 20 ng/mL rIL-3 and 50 ng/mL rSCF with or without 10 ng/mL rIL-4. The numbers of mast cells per culture were then counted on day 14.

  2. b)p<0.05 vs. corresponding value for cells cultured without rIL-4

1 × 104/culturerIL-3 + rSCF4.7±0.3
rIL-3 + rSCF + rIL-423.1±3.3b)+391
5 × 104/culturerIL-3 + rSCF55.6±4.8
rIL-3 + rSCF + rIL-40b)–100

These findings suggest that the IL-4-induced suppression of mast cell development may be dependent on the presence of other peritoneal cells of non-mast cell lineage. To evaluate this possibility further, we removed macrophages from these populations by pre-culturing peritoneal cells in plastic flasks several times before adding rIL-3, rSCF and rIL-4 to cultures of the remaining non-adherent cells. The suppressive activity of rIL-4 was greatly reduced after the incomplete removal of macrophages, however mast cell development in the presence of rIL-3 and rSCF was not affected. Replacing the macrophages completely restored the ability of the peritoneal cells to exhibit IL-4-induced suppression of mast cell development (Fig. 2A). To further confirm the role of macrophages, peritoneal cells from macrophage-deficient op/op mice were stimulated with rIL-3, rSCF and rIL-4. Compared to their littermate controls, mast cell development from peritoneal cells of op/op mice was not suppressed by IL-4 but was greatly promoted (Fig. 2B). These results demonstrate that IL-4-induced suppression of mast cell development requires the presence of macrophages.

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Figure 2. IL-4-induced suppression of mast cell development is dependent on the presence of macrophages. (A) Macrophages in the peritoneal cells of C57BL/6 mice were removed by pre-culturing the peritoneal cells in plastic flasks. The original peritoneal cells (PC), macrophage-depleted peritoneal cells (MΦ-PC) and macrophage-depleted peritoneal cells mixed with macrophages (MΦ-PC + MΦ) were cultured at a concentration of 2 × 105 cells/mL for 12 days for mast cell development. (B) Peritoneal cells from macrophage-deficient op/op and their littermate op/+ mice were cultured at a concentration of 1 × 105 cells/mL for 12 days for mast cell development. The cells were cultured in the presence of two cytokines (10 ng/mL rIL-3 and 50 ng/mL rSCF; hatched bars) or three cytokines (rIL-3, rSCF and 10 ng/mL rIL-4; black bars). Significant results (*p<0.05) compared to the corresponding values for MΦ-PC cultured with rIL-4 (A) and for peritoneal cells with op/op genotype cultured with rIL-4 (B) are indicated.

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Suppressive activity of supernatant collected from the cultures of IL-4-stimulated peritoneal cells

Next we tested whether IL-4 can stimulate macrophages to produce a soluble factor that can in turn suppress mast cell development. Purified mast cells or mast cell lines were cultured in supernatants collected from the cultures of peritoneal cells or peritoneal macrophages without nonadherent cells (such as T cells, B cells and NK cells). Compared to supernatants from cultures maintained with two cytokines (IL-3 and SCF), supernatants from cultures with three cytokines (IL-3, IL-4 and SCF) showed suppressive activity on IC-2 cells, MC/9 cells and purified mast cells (Table 3). Direct action of IL-4 was not responsible for the suppressive activity, since addition of a neutralizing antibody against IL-4 to the IL-4+ supernatants did not abolish their suppressive activity, and the addition of IL-4 to the IL-4 supernatants actually promoted the proliferation of mast cells and mast cell lines (data not shown). The IL-4+ supernatants from peritoneal cells of macrophage-deficient op/op mice did not show any suppressive activity on IC-2 cells, MC/9 cells or purified mast cells.

Table 3. Effects of supernatants collected from cultures of IL-4-stimulated peritoneal cells on the numbers of purified mast cells, IC-2 cells and MC/9 cells
Cells (5 × 104/culture)Viable cells/culture (× 104)a)Suppression (%)
IL-4 supernatantb)IL-4+ supernatantb)
  1. a) Purified mast cells, IC-2 cells and MC/9 cells were cultured in IL-4 supernatant or IL-4+ supernatant. Viable cells were counted on day 2.

  2. b) Peritoneal cells (5 × 105/mL) from normal C57BL/6 mice were cultured with two cytokines (10 ng/mL rIL-3 and 50 ng/mL rSCF) or three cytokines (rIL-3, rSCF and 10 ng/mL rIL-4). IL-4 and IL-4+ supernatants were then collected on day 8.

  3. c)p<0.05 vs. corresponding value for cells of the same type cultured in IL-4 supernatant

Purified mast cells25.0±3.43.3±0.4c)87
IC-211.4±3.01.9±0.2c)83
MC/914.7±0.91.6±0.3c)89

An apoptosis-inducing factor is responsible for the suppressive activity induced by IL-4

The above results demonstrate the presence of an IL-4-induced suppressive factor in the supernatants collected from the cultures of IL-4-stimulated peritoneal cells. Surprisingly, mast cells and immature mast cells underwent apoptosis when exposed to such supernatants despite the presence of high concentrations of mast cell growth factors, including IL-3, IL-4 and SCF (present in the tested culture supernatants at initial concentrations of 20, 10 and 50 ng/mL, respectively). Apoptosis was confirmed by observing the morphology of the cells by light microscopy, the redistribution of the membrane phospholipid phosphatidylserine (PS) of apoptotic cells from the inner face of the plasma membrane to the cell surface (Fig. 3A) and the DNA fragmentation of the apoptotic cells (Fig. 3B). These results indicate that an apoptosis-inducing factor is responsible for the IL-4-induced suppression of mast cell development and that mast cell growth factors such as IL-3, IL-4 and SCF are unable to effectively antagonize the apoptosis-inducing activity of the factor.

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Figure 3. Analysis of apoptosis. (A) FACS analysis of Annexin V-FITC-stained MC/9 cells. MC/9 cells were exposed to supernatants recovered from cultures of peritoneal cells stimulated with two cytokines (10 ng/mL rIL-3 and 50 ng/mL rSCF) or three cytokines (rIL-3, rSCF and 10 ng/mL rIL-4) for 24 h and were then stained with Annexin V-FITC for 5 min for FACS analysis (a: supernatant from a culture without rIL-4; b: supernatants from two separate cultures with rIL-4). (B) DNA fragmentation: Lane 1, complete DNA fragmentation of IC-2 cells after IL-3 deprivation for 24 h (positive control); Lanes 2 and 3, IC-2 cells cultured for 48 h in supernatants collected from cultures of peritoneal cells stimulated with two cytokines (rIL-3 and rSCF) or three cytokines (rIL-3, rSCF and rIL-4), respectively.

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IL-4-triggered production of the apoptosis-inducing factor depends on STAT6 signaling

To rule out the possibility that IL-4-induced production of the apoptotic factor is an artifact of the culture system, peritoneal cells from STAT6–/– and STAT6+/+ mice were cultured in the presence of rIL-3 and rSCF with or without rIL-4 or rIL-13. In contrast to the results for wild-type (STAT6+/+) mice, IL-4 and IL-13 failed to suppress mast cell development from the peritoneal cells of STAT6–/– mice (Fig. 4). Similar results were obtained when spleen cells, rather than peritoneal cells, were tested. Supernatants collected from cultures of IL-4- and IL-13-stimulated STAT6–/– peritoneal cells also failed to suppress the growth of purified mast cells or mast cell lines (data not shown).

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Figure 4. IL-4 and IL-13 do not suppress mast cell development from peritoneal cells of STAT6–/– mice. Peritoneal cells (105/mL) from STAT6+/+ (black bars) and STAT6–/– (hatched bars) mice were cultured in liquid medium containing 10 ng/mL rIL-3, 50 ng/mL rSCF and various concentrations of rIL-4 (A) or rIL-13 (B). Mast cells were counted on day 12 (*p<0.05 compared to the corresponding value for cells cultured without rIL-4 or rIL-13).

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Exclusion of Fas, FasL, perforin, TNF-α, TRAIL, TGF-β, IL-10, oncostatin M (OSM) or leukemia inhibitory factor (LIF) as the apoptosis-inducing factor

Fas/FasL and perforin are known to induce apoptosis of various cell types. In cultures of spleen and peritoneal cells from B6-lpr/lpr (Fas-deficient), B6-gld/gld (FasL-deficient) and perforin knock-out (PKO) mice, IL-4 was also able to suppress mast cell development (Fig. 5A). Supernatants from cultures of IL-4-stimulated peritoneal cells from the deficient mice also exhibited apoptosis-inducing activity against purified mast cells and mast cell lines (Fig. 5B). Furthermore, the apoptosis-inducing activity was not neutralized by antibodies against Fas/FasL, which were added to the supernatant before it was incubated with the target cells. These results demonstrate that Fas/FasL and perforin do not contribute significantly to the apoptosis-inducing activity in this system.

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Figure 5. Exclusion of Fas, FasL and perforin as the apoptosis-inducing factor. (A) Suppression of mast cell development from peritoneal cells of B6 wild-type (+/+), B6-lpr/lpr, B6-gld/gld or PKO mice by IL-4. Mouse peritoneal cells (105/mL) were cultured in the presence of various cytokines (10, 50 and 10 ng/mL for rIL-3, rSCF and rIL-4, respectively). Mast cells were counted on day 10. (B) Suppression of growth of purified mast cells by supernatants recovered from the cultures of IL-4-stimulated peritoneal cells of B6-+/+, B6-lpr/lpr, B6-gld/gld or PKO mice. Peritoneal cells (5 × 105/mL) from various mice were cultured in medium containing rIL-3 and rSCF with or without rIL-4. IL-4 and IL-4+ supernatants were then collected on day 8. Purified mast cells (105/mL) developed from normal C57BL/6 mouse peritoneal cells were exposed to the IL-4 or IL-4+ supernatants. The viable cells were then counted on day 3 (*p<0.05 compared to the corresponding value for cells of the same genotype cultured without rIL-4).

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The possibility that the apoptosis-inducing factor may be IL-10, TNF-α, TGF-β, LIF or OSM was excluded by testing rIL-10, rTNF-α, rTGF-β1 and rOSM as well as neutralizing antibodies against IL-10, TNF-α, TGF-β1+2 and LIF. Instead of inducing suppression, both IL-10 and TNF-α promoted mast cell development from mouse spleen cells 21, 25. By RT-PCR analysis, it was confirmed that IL-4 did not significantly stimulate the expression of TRAIL mRNA, and the neutralizing antibody against TRAIL did not abolish the apoptosis-inducing activity of the recovered supernatants. TGF-β partially antagonized the activities of IL-3 and SCF in mast cell development and growth (Table 4). However, neither mast cell lines nor purified mast cells underwent apoptosis after exposure to rTGF-β. Moreover, a neutralizing antibody against TGF-β did not eliminate the apoptosis-inducing activity of the supernatants recovered from the cultures of IL-4-stimulated peritoneal cells.

Table 4. Effect of TGF-β on mast cell development and mast cell numbers
CellsCytokinesa)Mast cells or viable cells per culture (× 104)Suppression (%)
  1. a) Cytokines: 20 ng/mL rIL-3, 50 ng/mL rSCF, 10 ng/mL rIL-4 and 5 ng/mL rTGF-β

  2. b) Peritoneal cells (105/mL/well) of normal C57BL/6 mice were cultured in liquid medium with various cytokines. Mast cells were counted on day 12.

  3. c) Purified mast cells (105/mL/well) were cultured with various cytokines, and viable cells were counted on day 3.

  4. d)p<0.05 vs. value for cells of the same type cultured without rIL-4 and/or rTGF−β

Peritoneal cellsb)rIL-3 + rSCF86.2±2.5
rIL-3 + rSCF + rIL-40d)100
rIL-3 + rSCF + rTGF-β50.0±0.5d)42
Purified mast cellsc)rIL-3 + rSCF + rIL-498.7±12.7
rIL-3 + rSCF + rIL-4 + rTGF-β70.4±16.3d)29

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Although IL-4 can function as a growth factor for mast cells, our previous work showed that IL-4 is also able to inhibit mast cell development from mouse spleen cells 21. In this study, we have found that down-regulation of mast cell development appears to be a general function of IL-4, since IL-4 dose-dependently suppressed mast cell development from peritoneal and bone marrow cells as well as from spleen cells (Fig. 1). The possibility that IL-4 may promote the growth of mast cells but inhibit the growth of mast cell precursors was excluded, because IL-4 synergistically promoted the activities of IL-3 and SCF on both mast cells and mast cell precursors (Table 1).

IL-4 also displayed contradictory activities if different densities of peritoneal cells were cultured. When only 104 peritoneal cells were seeded per culture, IL-4 dramatically promoted mast cell development. In contrast, when 5 × 104 or more peritoneal cells were added per culture, mast cell development was completely suppressed by IL-4 (Table 2). These results suggest that non-mast cell lineage cells in the cultures are crucial for IL-4-induced suppression of mast cell development. This hypothesis was confirmed by experiments in which we removed peritoneal macrophages from, and in some cases returned them to, the cultures (Fig. 2A) and by experiments using peritoneal cells from macrophage-deficient op/op mice (Fig. 2B). Thus, IL-4-induced suppression of mast cell development is an indirect process that is dependent on an apoptosis-inducing factor produced by macrophages (Fig. 3, Table 3 and Fig. 5B).

Both mast cells and mast cell precursors underwent apoptosis when exposed to supernatants collected from the cultures of IL-4-stimulated peritoneal cells or purified peritoneal macrophages (Fig. 3, Fig. 5B and Table 3), indicating the presence of an apoptosis-inducing factor in these cultures. The factor is a protein rather than a lipid or polysaccharide. This was confirmed by pretreatment of the supernatant with a large amount of trypsin (1 mg/mL) overnight at 37°C; the trypsin-digested supernatant lost apoptosis-inducing activity. The molecular weight of the apoptosis-inducing factor was estimated by gel filtration of the IL-4-stimulated supernatant and comparison with molecular markers to be 35 to 45 kDa.

STAT6 is a signal transduction molecule that is activated only by IL-4 and IL-13, which share a receptor chain 26. IL-4-induced production of the apoptosis-inducing factor is dependent on STAT6 signaling, because both IL-4 and IL-13 failed to suppress mast cell development from the peritoneal cells of STAT6–/– mice (Fig. 4A, B), and the supernatant collected from cultures of IL-4- and IL-13-stimulated STAT6–/– peritoneal cells did not exhibit the apoptosis-inducing activity. In contrast to the IL-4- and IL-13-triggered production of the apoptosis-inducing factor via a STAT6-dependent mechanism, it seems that the promotion of mast cell proliferation by IL-4 and IL-13 is independent of STAT6 signaling. In this way, mast cell development from the peritoneal cells of STAT6–/– mice was dose-dependently promoted by IL-4 and IL-13 (Fig. 4A, B). This result is in agreement with a prior report showing that STAT6 is not essential for IL-4-mediated proliferation of mouse mast cells 27.

Human rIL-4 has been reported to induce apoptosis of cord blood-derived mast cells but not lung- and fetal liver-derived mast cells. This process was considered to be the result of a direct effect of IL-4, because highly purified cord blood-derived mast cells also underwent apoptosis after exposure to human rIL-4 24, 28. In mice, IL-4 has been reported to elicit apoptosis of developing mast cells from bone marrow cells directly, via a mitochondrial pathway 23. Moreover, the combination of IL-4 and IL-10 also directly induces apoptosis in IL-3-dependent bone marrow-derived mast cells and peritoneal mast cells 22.

In our system, however, direct effects of IL-4, alone or in combination with IL-10, on mast cell apoptosis can be completely excluded. Removal of IL-4 and/or IL-10 from the supernatant collected from the cultures of IL-4-stimulated peritoneal cells by neutralizing antibodies did not abolish the apoptosis-inducing activity. Moreover, instead of inducing suppression, IL-4 promoted the proliferation of spleen-derived or peritoneal mast cells as well as immature (IC-2) mast cells (Table 1 and 2). IL-10 has also been shown to promote mast cell development from mouse spleen cells 21.

It has been reported that mast cells express Fas (CD95) antigen on their surface and that an antibody against Fas can induce mast cell apoptosis 29. Perforin can also play an important role in cell death and apoptosis 30. To assess the possible involvement of Fas/FasL and perforin in our system, spleen and peritoneal cells from Fas/FasL-deficient and PKO mice were cultured with IL-3, SCF and IL-4 and then examined for mast cell development. The data demonstrate that Fas/FasL and perforin are not related to the apoptosis-inducing activity (Fig. 5). This conclusion was further confirmed by tests with neutralizing antibodies against Fas and FasL.

TGF-β is known to be a suppressive factor of various types of cells 31 and can influence mast cell apoptosis 32. In the current study, TGF-β did weakly antagonize the activities of IL-3 and SCF on mast cell growth and development (Table 4). However, under the conditions tested in our experiments, mast cell lines and purified mast cells did not undergo apoptosis after exposure to TGF-β. Also, a neutralizing antibody against TGF-β did not eliminate the apoptosis-inducing activity of supernatants collected from the cultures of IL-4-stimulated peritoneal cells. Using other neutralizing antibodies as well as recombinant cytokines, we also showed that the apoptosis-inducing factor is not TNF-α, TRAIL, OSM or LIF.

By affecting the numbers of mast cells in normal tissues or at sites of pathology, factors that regulate mast cell proliferation and/or differentiation may significantly influence physiological processes or diseases, especially in relation to Th2- and IgE-associated hypersensitivity reactions and allergic disorders. However, most research on mast cell development has largely focused on mast cell growth factors and/or on changes in mast cell populations during Th2-associated responses. Our studies have characterized a novel IL-4-dependent mechanism that can contribute to mast cell homeostasis and/or to changes in mast cell populations during Th2-associated responses. The fact that IL-4 induces the production of this factor from macrophages offers some insight into why IL-4 can promote mast cell growth under certain circumstances (particularly in the absence of other cell types) but can promote mast cell apoptosis and suppress mast cell development in other settings. The molecular identification of this mast cell apoptosis-inducing factor may be of considerable interest.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Mice

Male C57BL/6N (B6), C3H/HeN and BALB/c mice (5–10 wks old) were purchased from Charles River Japan (Atsugi, Japan). B6 wild-type (B6-+/+), B6-lpr/lpr, B6-gld/gld, C57BL/6 -PFPtm1Sdz (PKO), C. 129S2-STAT6 (tm1Gru) (STAT6 knock-out) and B6C3Fe a/a-Csf1<op>/J mice were purchased from The Jackson Laboratory (Bar Harbor, ME). The macrophage colony stimulating factor (M-CSF)/macrophage-deficient osteopetrotic (op/op) and littermate control (+/op or +/+) genotypes were raised in our laboratory from breeding pairs of the mice. The op/op mice were characterized by failure of incisor eruption and were fed with a granulated diet. The experiments were approved by the Animal Research Ethics Board of Showa University.

Cell lines

IC-2 (mast cell precursor) and MC/9 (mast cell) lines were supplied by Cell Bank (Riken BioResource Center, Tsukuba, Ibaraki, Japan). Both IC-2 and MC/9 are dependent on IL-3 or SCF for survival and proliferation 3335.

Reagents

The following reagents were purchased from the indicated companies: murine rIL-3 (Intergen Company, NY, NY); rIL-4, rIL-10, rIL-13, rSCF, rTNF-α and OSM as well as goat anti-mouse IL-3, IL-4, IL-10, TNF-α and LIF neutralizing Ab (R&D Systems, Minneapolis, MN); purified rat anti-mouse TRAIL neutralizing mAb, hamster anti-mouse Fas [Cluster of Differentiation 95 (CD95)] and Fas ligand (FasL, CD95 ligand) mAb (PharMingen, San Diego, CA); human rTGF-β1 and neutralizing rabbit anti-human TGF-β1+2 antiserum (King Brewing Co. Ltd, Central Institute, Kakogawa-shi, Japan).

Mast cell development from mouse spleen, peritoneal and bone marrow cells

Mast cells were generated from mouse spleen cells as described previously 21, 3638. Briefly, spleen cells (6 × 106 to 10 × 106/mL) were cultured for 12 days in RPMI 1640 medium (Life Technologies, Grand Island, NY) containing 10% FCS, 25 mM Hepes, 200 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin and 5 × 10–5 M 2-ME (complete culture medium). The cultures (1 mL/well) were incubated in 24-well plates at 37°C in a humidified atmosphere with 5% CO2. One-half of the volume of the culture medium was changed every 4 days. Various stimulants or cytokines were added at the start of the culture to assess their effects on mast cell development. Mast cell numbers were counted on day 12. FCS used in this system was strictly selected according to the level of contaminating LPS (less than 0.05 ng/mL) and was confirmed to be non-mast cell-inducing sera 37.

Mast cells were also generated from peritoneal exudate cells and bone marrow cells. Peritoneal or bone marrow cells (105/mL) were cultured in complete culture medium containing 10 ng/mL rIL-3 and 50 ng/mL rSCF with or without 10 ng/mL rIL-4. The medium was changed with replenishment of cytokines every 4 days. Mast cell numbers were counted on day 12. To obtain mast cells of >90% purity (referred to in the text as “purified mast cells”), peritoneal cells were cultured in the presence of 10 ng/mL rIL-3 and 50 ng/mL rSCF for 16 to 20 days, and dead cells were removed by Histopaque-1077 (Sigma Chemical Co., St. Louis, MO) separation.

Mast cell colony formation in semi-solid culture

Methylcellulose cultures were used for an in vitro colony formation assay of mast cells as described previously 38. Peritoneal cells and c-kit+ cells separated from spleen cells were suspended in 1 mL complete culture medium containing 1% methylcellulose (Sigma), 20 ng/mL rIL-3 and 50 ng/mL rSCF with or without 10 ng/mL rIL-4. The cells were added to a 6-well culture plate, each well with a diameter of 35 mm, and were maintained at 37°C in a humidified atmosphere with 5% CO2 for 14 days. The cells were then collected, and mast cell numbers were counted using Alcian blue staining.

Separation of c-kit+ cells by MACS

The c-kit+ cells among splenocytes were separated using a MACS column according to the manufacturer's instructions. Briefly, spleen cells were incubated with rat anti-mouse CD117 (c-kit) mAb (IgG2b, PharMingen) at 4°C for 30 min. After washes, the cells were further incubated with goat anti-rat IgG Ab conjugated to super-paramagnetic microbeads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) at 4°C for 15 min. The cells were washed again and then applied to the MACS column. The positive fraction was collected and cultured for assessment of mast cell development.

Collection of supernatants from mouse peritoneal cell cultures

Peritoneal cells (5 × 105/mL) from various mice were cultured in complete culture medium containing two cytokines (20 ng/mL rIL-3 and 50 ng/mL rSCF) or three cytokines (rIL-3, rSCF and 10 ng/mL rIL-4). The IL-4 and IL-4+ supernatants were then collected on day 8 and used as culture media to evaluate the IL-4-triggered production of a factor that can induce apoptosis in IC-2, MC/9 and purified mast cells.

Mast cell and viable cell counts

The cultures were performed in triplicate, and the numbers of cells are expressed as mean ± SD. Mast cells were counted using an Alcian blue staining method specific for counting mast cells and basophils 39. Viable cells were counted using trypan blue exclusion after IC-2, MC/9 and purified mast cells were cultured in the supernatants containing the apoptosis-inducing factor for 2 or 3 days.

DNA fragmentation assay

DNA fragmentation was detected by agarose gel electrophoresis. IC-2, MC/9 and purified mast cells were cultured for 2 days in the supernatant from peritoneal cells incubated with or without rIL-4. DNA was then extracted from the cells for electrophoresis. The DNA from IC-2 cells cultured for 2 days after withdrawal of IL-3 was used as a positive control for DNA fragmentation.

Annexin V staining of apoptotic cells

Apoptosis was also detected using an Apoalert Annexin V Apoptosis Kit (CLONTECH Laboratories, Palo Alto, CA), which is based on redistribution of the membrane PS of apoptotic cells from the inner face of the plasma membrane to the cell surface. IC-2 cells were cultured for 24 h in supernatants from peritoneal cells that had been stimulated with or without rIL-4. The cells were incubated for 5 min with 1 μg/mL Annexin V-FITC, which binds to surface PS, and the cells were then analyzed using a fluorescence-activated cell sorter (FACStar, Becton Dickinson, Bedford, MA).

RT-PCR analysis of TRAIL expression

Total RNA was extracted from peritoneal cells that had been cultured with or without rIL-4 for 2 days. cDNA synthesis and RT-PCR were performed using the ProSTAR HF Single Tube RT-PCR System (Stratagene, La Jolla, CA). The sense and anti-sense primers used for PCR of TRAIL were 5′-CTGGACAGCAGTAGTCCTTC-3′ and 5′-GGGCTGTGTTTGATCTTTAC-3′. Thirty amplification cycles were used, each including a denaturation step for 30 s at 95°C, an annealing step for 30 s at 60°C and an extension step for 30 s at 72°C. PCR products were analyzed on 1.2% agarose gels and visualized using ethidium bromide staining.

Estimation of the molecular weight of the apoptosis-inducing factor by gel filtration

The recovered supernatant from IL-4-stimulated peritoneal cells was first dialyzed against PBS and then concentrated using an Advantec UK-10 ultrafiltration membrane (Toyo Roshi Kaisha, Japan) with a nominal cutoff of 10 kDa. Concentration of the supernatant was carried out in an ice bath under 15 psi N2. Approximately 3 mL of the supernatant concentrated four times by this membrane was then loaded onto a Bio-Gel A 0.5 column (1.5 × 100 cm, Pharmacia, Uppsala, Sweden), which was equilibrated with PBS (pH 7.2). The flow rate was approximately 10 mL/h, and the volume collected was 2 mL/tube. The apoptosis-inducing activity of the fractions eluted from the Bio-Gel column was determined. The molecular weight of the fraction with the apoptosis-inducing activity was estimated by comparing the fractions of molecular markers eluted from the same column under the same conditions.

Presentation of data and statistical analyses

All experiments were performed three or more times to confirm their repeatability. Data from control and experimental groups were compared by the Student's t-test. Values of p<0.05 were considered statistically significant. The percent promotion or suppression of mast cell growth and development by IL-4 was calculated as follows: % Promotion or % Suppression = (number of mast cells in culture with IL-4 / number of mast cells in culture without IL-4 –1) ×100. The + and – signs indicate promotion and suppression, respectively.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

This work was supported by grants No. AI23990, CA72074 and HL67674 from The National Institutes of Health, USA (to S. J. G.) and No. 13670477 from the Ministry of Education, Culture, Sports, Science and Technology, Japan (to Z.-Q. H.).

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