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. 2009 Oct;23(10):3506–3515. doi: 10.1096/fj.08-128900

Sphingosine-1-phosphate induces development of functionally mature chymase-expressing human mast cells from hematopoietic progenitors

Megan M Price 1, Dmitri Kapitonov 1, Jeremy Allegood 1, Sheldon Milstien 1, Carole A Oskeritzian 1, Sarah Spiegel 1,1
PMCID: PMC3236593  PMID: 19535686

Abstract

Mast cells (MCs) play a critical role in both acute and chronic inflammation and mature in peripheral tissues from bone marrow-derived progenitors that circulate in the blood as immature precursors. MCs developed from cord blood-derived progenitors cultured with stem cell factor (SCF) alone express intragranular tryptase (MCTs), the phenotype predominant in the lung. MC progenitors are likely to encounter the serum-borne bioactive sphingolipid metabolite, sphingosine-1-phosphate (S1P), during migration to target tissues. S1P accelerated the development of cord blood-derived MCs (CB-MCs) and strikingly increased the numbers of MC-expressing chymase. These MCs have functional FcεRIs, and similar to skin MCTCs that express both tryptase and chymase, also express CD88 and are activated by anaphylatoxin C5a and the secretagogue compound 48/80. S1P induced release of IL-6, a cytokine known to promote development of functionally mature MCTCs, from cord blood cultures containing adherent macrophages, and from highly purified macrophages, but not from macrophage-depleted CB-MCs. In contrast, S1P stimulated secretion of the chemokine, monocyte chemoattractant protein 1 (MCP-1/CCL2), from these macrophage-depleted and purified CB-MCs. These results suggest crucial roles for S1P in regulating development of human MCs and their functions and reveal a complex interplay between macrophages and MC progenitors in the development of mature human MCs.—Price, M. M., Kapitonov, D., Allegood, J., Milstien, S., Oskeritzian, C. A., Spiegel, S. Sphingosine-1-phosphate induces development of functionally mature chymase-expressing human mast cells from hematopoietic progenitors.

Keywords: tryptase, anaphylatoxin, CCL2, S1P receptors, degranulation, IL-6

Mast cells (MCs) are key effector cells involved in orchestrating and perpetuating inflammatory responses. They are tissue-dwelling cells derived from hematopoietic stem cells that circulate in the blood as committed progenitors until they enter the tissues to complete their maturation (1). Once mature, MCs reside in the perivascular spaces of all tissues and contain intracytoplasmic granules rich in acidic proteoglycans. There are two subpopulations of human MCs based on the composition of their intragranular protease repertoire: those expressing tryptase only (MCTs) resemble mucosal MCs and are predominant in lung; those that contain chymase in addition to tryptase (MCTCs) are similar to connective tissue MCs and the phenotype of skin MCs (2, 3). Stem cell factor (SCF), the Kit ligand, is an important growth factor required for MC survival and differentiation and is the only growth factor identified so far that by itself in vitro causes human hematopoietic progenitor cells to become tryptase-producing MCs (4, 5). Several cytokines, including IL-3, IL-4, IL-5, IL-6, and IL-9, enhance the mitogenic effects of SCF on cord blood-derived cultured human MCs (CB-MCs) in vitro, and some of them are also cytoprotective (6,7,8,9). Much less is known of how human hematopoietic progenitor cells differentiate into mature MCTCs and the factors that influence chymase expression. A notable exception is IL-6, which induces chymase protein expression in SCF-dependent CB-MCs that normally only express tryptase (10,11,12,13).

Sphingosine-1-phosphate (S1P) is a potent lipid mediator produced and secreted by activated MCs to regulate their functions (reviewed in refs. 14, 15). Similar to crosslinking of the high-affinity IgE receptor (FcεRI), SCF also activates both isoforms of sphingosine kinase (SphK1 and SphK2) in MCs, leading to S1P formation (16). It has been suggested that SphK2 is required in murine MCs for production of S1P, cytokine secretion, and degranulation. However, susceptibility of mice to in vivo anaphylaxis correlated with circulating S1P generated by SphK1 from a non-MC source (17). MCs express two of the five known S1P receptors, S1P1 and S1P2, and activation of these receptors by secreted S1P is important in movement of rodent MCs and their degranulation, respectively (18, 19).

MC precursors circulate in the blood, where they have the opportunity to encounter various serum-borne growth factors, including S1P. Lysophosphatidic acid (LPA), another phospholipid mediator present in serum that is structurally related to S1P was shown to increase the number of cord blood-derived mast cells (CB-MCs) (20). Given that S1P is also present in human serum at high nanomolar concentrations (21) and can influence MC responses (14, 22), it was of interest to examine the involvement of S1P in development of MCs derived from human hematopoietic progenitors. Remarkably, S1P increased the number of CB-MCs and strikingly increased chymase expression and CD88, the receptor for C5a. Our results also reveal that cooperation between monocytes/macrophages and MC progenitors may be important for the development of mature chymase expressing MCs.

MATERIALS AND METHODS

Reagents

S1P was obtained from Biomol (Plymouth Meeting, PA, USA). VPC23019 was obtained from Avanti (Alabaster, AL, USA). JTE-013 was obtained from Tocris (Ellisville, MO, USA). SCF was a generous gift from Amgen (Thousand Oaks, CA, USA). Recombinant human IL-6 was purchased from R&D Systems (Minneapolis, MN, USA). Anti-tryptase and anti-chymase monoclonal antibodies (mAbs) were obtained from Chemicon (Temecula, CA, USA). Dinitrophenyl-human serum albumin (DNP-HSA, Ag), C5a, and compound 48/80 were from Sigma-Aldrich (St. Louis, MO, USA), and anti-human IL-6 mAb was from Invitrogen (Carlsbad, CA, USA).

Culture of human CB-MCs

Umbilical cord blood was obtained at the time of delivery and collected in heparin-treated tubes. The experimental protocol was approved by the Institutional Review Board at Virginia Commonwealth University. Cord blood was diluted and overlaid on Histopaque (density 1.077 g/ml) and then centrifuged to remove erythrocytes. Mononuclear cells at the plasma-Histopaque interface were collected, washed, and subjected to a second Histopaque density gradient centrifugation (10). Purified mononuclear cells were cultured in 24-well plates at 5 × 105 cells/ml in RPMI 1640 containing heat-inactivated controlled process serum replacement medium (CPSR-3, Sigma), 2 mM l-glutamine, 0.1 mM nonessential amino acids, 10 mM HEPES (pH 7.2), 50 μM 2-mercaptoethanol, 200 U/ml penicillin, 100 μg/ml streptomycin, and 100 ng/ml SCF (10) in the absence or presence of S1P, as indicated in figure legends. Culture medium with indicated supplements was replaced weekly. Slides were stained with Toluidine blue to assess metachromasia and MC numbers. Cell numbers and viability (always >80% as determined by Trypan blue exclusion) were assessed immediately prior to experiments.

Immunomagnetic purification of CB-MCs by negative depletion of CD14-positive cells

Monocytes/macrophages were immunodepleted from cultures using anti-CD14-coated magnetic Dynabeads (4 beads/target cell; Dynal Biotech, ASA, Oslo, Norway), essentially as recommended by the manufacturer, except for the omission of sodium citrate/EDTA. Unattached CD14-negative cells (MCs) were collected and cultured as described previously (23), and contained 95–99% MCs, as determined by Toluidine blue staining.

Preparation of macrophages from cord blood cultures

Macrophages were highly enriched by positive selection with biodegradable anti-CD14-MicroBeads using a SuperMACS (Miltenyi Biotec, Auburn, CA, USA), according to the manufacturer’s protocol. Ninety to 95% of the cells were positive for CD14 expression by immunofluorescence analysis.

Immunofluorescence and immunocytochemistry

CB-MCs (5×104)were smeared onto glass slides, fixed in methanol containing 0.6% H2O2 for 30 min at room temperature, and stored at −80°C. Slides were incubated with 10 μg/ml tetramethylrhodamine isothiocyanate-conjugated anti-tryptase G3 mouse mAb (G3-TRITC) or isotype-matched negative control (MOPC-TRITC) for 1 h at 37°C, washed three times in 0.01 M Tris-buffered saline (pH 7.4) containing 0.05% Tween 20 (TBST). Cells were visualized by fluorescence microscopy with a Nikon TE300 (Nikon, Tokyo, Japan), and the percentage of positively stained cells was calculated. At least 200 cells were scored in a double-blinded manner. Images were also collected with a Zeiss LSM 510 Meta confocal microscope (Carl Zeiss, Oberkochen, Germany) with the optical slice set to 1 μm for all channels. All images were exported directly using Zeiss LSM Image Examiner (v. 3.2.0.70) to 8-bit TIFF files without compression, contrast, or gamma adjustments.

Slides were stained for chymase with biotin-conjugated anti-chymase B7 mAb (B7-B) or isotype-matched negative control (MOPC-B) overnight at 4°C, as described previously (10). Briefly, slides were washed in TBST and incubated with streptavidin-peroxidase conjugate (20 μg/ml) for 1 h at room temperature. After washing, slides were incubated with 3-amino-9-ethylcarbazole in 0.01% H2O2 for 7 min at room temperature, and chymase-positive MCs were identified by brown staining. Slides were examined with a Nikon Eclipse E800 microscope equipped with an ×100 objective, and the percentage of positively stained cells was calculated. At least 200 cells were scored in a double-blinded manner. In some experiments, after washing, slides were incubated with alkaline phosphatase-conjugated antitryptase G3 mAb (10 μg/ml) at 4°C. Slides were then washed and incubated with SigmaFast Fast Red TR/Naphthol AS-MX phosphate (4-chloro-2-methylbenzenediazonium/3-hydroxy-2-naphthoic acid 2,4-dimethylanilide phosphate; Sigma), and tryptase-positive MCs were identified by pink-red staining.

Degranulation

CB-MCs were sensitized with 1 μg/ml anti-DNP IgE overnight, washed once to remove unbound IgE, and then stimulated without or with DNP-HSA (Ag, 30 ng/ml), as described previously (19). Degranulation was determined by measuring the release of the granule marker, β-hexosaminidase, with a colorimetric enzyme assay (18). Values are expressed as the percentage of total cellular β-hexosaminidase released into the medium. Spontaneous degranulation of unstimulated cells was <10%.

Flow cytometry

To determine expression of surface CD88, CB-MCs were incubated with rabbit anti-human CD88 mAb (10 μg/ml) or a nonimmune rabbit IgG (10 μg/ml) as a negative control, followed by staining with Alexa Fluor 488-labeled goat anti-rabbit IgG (5 μg/ml) (Molecular Probes, Eugene, OR, USA) (23). After staining, the cells were washed once with PBS and resuspended in FACS buffer. Flow cytometric analysis was performed using the FC500 combined with CXP software (Beckman Coulter, Fullerton, CA, USA).

ELISA

Human IL-6 and CCL2/MCP-1 were measured by ELISAs with purified biotinylated mouse or rat mAbs specific for each cytokine. Standard curves were prepared with recombinant cytokines (BD Biosciences, San Diego, CA, USA). Assays were performed in Maxisorb 96-well plates (Nunc, Roskilde, Denmark), according to the manufacturer’s protocols. Briefly, wells were coated overnight at 4°C with capture mAbs, blocked with PBS containing 10% FBS, washed in PBS containing 0.05% Tween 20, and incubated for 2 h at room temperature with standards or samples diluted in PBS with 10% FBS. Wells were washed, incubated with biotin detection mAbs, and streptavidin-HRP conjugate for 1 h at room temperature, washed, and incubated with peroxidase substrate. Absorbance was measured at 450 nm with an EL800 microplate reader (Biotek, Winooski, VT, USA). The lower limits of detection for IL-6 and MCP-1/CCL2 were 4.7 and 7.8 pg/ml, respectively.

Quantitation of S1P

Lipids were extracted from medium and cells, and S1P was measured by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS; 4000 QTRAP; ABI, Foster City, CA, USA), as described previously (24).

Statistical analysis

Experiments were repeated at least 3 times with consistent results. For each experiment, data from triplicate samples were calculated and expressed as means ± sd. Differences between groups were determined with the paired Student’s t test, where P ≤ 0.05 was considered significant.

RESULTS

S1P induces development of cord blood progenitor cells to chymase-expressing MCs

MC precursors circulate in the blood, where they have the opportunity to encounter various serum-borne growth factors, including S1P (21, 25). Therefore, we sought to examine whether S1P might influence the development of MCs derived from human hematopoietic progenitors. To this end, S1P was added to cord blood mononuclear progenitors cultured in chemically defined medium containing human SCF, a growth factor that is able to induce hematopoietic progenitor cells cultured in vitro to become MCs (5). The addition of 1 μM S1P, the concentration found in normal human plasma (26), had no significant effects on viability of cord blood mononuclear cultures compared to cells cultured in the presence of SCF alone. To examine the effects on development of MCs, cultures were immunostained for tryptase, an intragranular marker of MCs. After prolonged culture in the presence of S1P, a higher proportion of cells stained positively for tryptase compared to cultures with SCF alone (Fig. 1A). Although coculture of progenitors with SCF and S1P for 3 wk had no significant effects on the proportion of tryptase-positive cells or on metachromasia (Fig. 1B), by 6 to 7 wk, 1 μM S1P increased the proportion of tryptase-positive cells compared to cells cultured with SCF alone. Similarly, after 8 wk of culture with concentrations of 0.1 and 1 μM S1P, there was an enhancement of numbers of tryptase-positive MCs of 1.6- and 3-fold, respectively (Fig. 1A, C). Lower concentrations of S1P did not show consistent effects.

Figure 1.

Figure 1

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Effect of S1P on tryptase expression in developing hMCs. A, B) Cord blood mononuclear cells were cultured for the indicated weeks with SCF (100 ng/ml) alone (none) or in the absence (vehicle) or presence of S1P (0.1 and 1 μM). Cultures were stained with anti-tryptase mAb (G3-TRITC) to assess tryptase expression, and tryptase-positive cells were quantified as described in Materials and Methods. A minimum of 200 cells was scored in a double-blind manner. Data are expressed as total number of tryptase-positive cells (A). Results from a cord blood culture from another donor are expressed as percentage of tryptase-positive cells (B). Data are means ± sd. *P < 0.05, **P < 0.01 vs. untreated controls. C) Representative fields of cells from a different donor stained with G3-TRITC and corresponding DIC images after treatment without (vehicle) or with S1P (0.1 μM) for 8 wk. Scale bars = 50 μm. Similar results were obtained with two additional cord blood cultures from other donors.

In agreement with previous studies (10, 13, 20), human progenitor cells cultured in the presence of SCF alone only expressed tryptase and had no detectable chymase in their granules, as determined by immunocytochemistry (Fig. 2A, C). Remarkably, culturing in the presence of S1P not only increased the number of MCs but strikingly increased chymase expression (Fig. 2A–C). A small increase was detected within 4 wk of culture in the presence of 1 μM S1P (Fig. 2A). However, after 6 wk of culture, even a concentration of S1P as low as 0.1 μM induced a significant increase in chymase-positive MCs (Fig. 2A, B). Tryptase staining in the granules of cells cultured for 8 wk in the presence of S1P was observed by confocal microscopy (Fig. 2E). These cells also contained granules that stained strongly with Toluidine blue (Fig. 2F). Immunocytochemistry of these S1P-treated MCs revealed strong chymase staining compared to cells cultured with SCF alone (Fig. 2C, G). Moreover, as expected, the chymase expressing MCs also expressed tryptase (Fig. 2H). These results are reminiscent of many previous studies showing that after culturing cord blood mononuclear cells in the presence of SCF and IL-6 for 8 wk, all MCs had tryptase-positive granules, while ∼20% also expressed chymase (6, 8, 27).

Figure 2.

Figure 2

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S1P induces chymase expression in developing hMCs. A–C) Cord blood mononuclear cells were cultured for the indicated weeks with SCF (100 ng/ml) alone (none) or in the absence (vehicle) or presence of the indicated concentrations of S1P. Cells were stained with anti-chymase mAb or negative control IgG, and chymase-positive cells were quantified as described in Materials and Methods. A minimum of 200 cells was scored in a double-blind manner. A) Data are expressed as total number of chymase-positive cells. B) Results from a cord blood culture from another donor are expressed as percentage of chymase-positive cells. Negative control staining was <1%. Similar results were obtained with two additional cord blood cultures from different donors. Data are expressed as means ± sd. *P < 0.05, **P < 0.01 vs. untreated controls. C) Cells from a different donor photographed under light microscopy at ×200 magnification after treatment without (vehicle) or with S1P (0.1 μM) for 10 wk. D–H) Histochemical characteristics of CB-MCs cultured in the presence of S1P. Cord blood mononuclear cells were cultured for 8 wk with SCF and S1P (0.1 μM). D, E) Cells were visualized by confocal microscopy for DIC (D) and tryptase staining (E). Scale bars = 5 μm. F, G) Toluidine blue (F) and chymase (G) staining were visualized by light microscopy. H) Cells were stained for both chymase and tryptase. Representative individual cells from 3 experiments are shown. I–K) CD88 cell surface expression. Cord blood mononuclear cells were cultured for 8 wk with SCF in the absence (J) or presence of 1 μM S1P (I, K) and stained with rabbit anti-CD88 (J, K) or with nonimmune rabbit IgG (I) followed by staining with Alexa Fluor 488-labeled secondary antibody and sorted by flow cytometry. Sorting gates are indicated.

In agreement with previous studies showing that surface CD88, the receptor for C5a (C5aR), is only expressed by MCTCs (23, 28), there was no detectable expression of CD88 on CB-MCs cultured for 8 wk in the presence of SCF alone (Fig. 2J), whereas in the presence of 1 μM S1P, 13% of MCs expressed CD88 on the cell surface, as determined by FACS analysis (Fig. 2K). This result is consistent with the observation that 13.3% of the cells in this MC culture exposed to S1P are also chymase positive at that time (Fig. 2B). Although similar levels of chymase expression were found in cultures from three donors, 29% and 35% of the MCs from two other donors were chymase positive after culturing with 1 μM S1P for 8 wk. This is the first demonstration that a serum-borne bioactive lipid can induce differentiation of CB-MC progenitors to mature chymase expressing MCs.

S1P-induced secretion of IL-6 from cord blood cultures is dependent on the presence of monocytes/macrophages

As IL-6 induces chymase protein expression in SCF-dependent CB-MCs (10,11,12,13), it was of interest to determine whether the effect of S1P on chymase expression in developing MCs was due to production and secretion of IL-6. In agreement with previous studies (13, 23), IL-6 was not detected in supernatants of MCs cultured for 1 wk in the presence of SCF alone. However, cultures also treated with S1P secreted small amounts of IL-6 (Fig. 3A). As secreted IL-6 is not stable for a period of 1 wk (29), we next measured its secretion during the 24-h period after S1P addition to the culture medium. Interestingly, treatment with S1P induced secretion of significant amounts of IL-6 as early as 6 h compared to cultures treated with SCF alone in the absence or presence of vehicle (Fig. 3B). Maximum IL-6 accumulation in the medium was observed at 10 h following addition of S1P and declined thereafter, consistent with its degradation by MC-derived proteases (29). However, it should be noted that levels of IL-6 were still significantly elevated even 24 h after the addition of S1P.

Figure 3.

Figure 3

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S1P enhances IL-6 secretion from cultures of cord blood-derived progenitors but not from purified CB-MCs. A) Cord blood progenitors were cultured for 8 wk with SCF (100 ng/ml) alone (open bar) or in the presence of S1P (0.1 and 1 μM, solid bars). Cells (106) were then stimulated again with SCF in the absence or presence of S1P for 1 wk, and IL-6 in supernatants was measured by ELISA. *P < 0.01 vs. vehicle treatment. B) Cord blood progenitors were cultured for 8 wk with SCF (100 ng/ml) alone (none; circles), or in the presence of vehicle (triangles) or 1 μM S1P (squares). Cells (106) were then stimulated again for the indicated times, and IL-6 in supernatants was measured by ELISA. C, D) After 8 wk in culture with SCF alone, purified CB-MCs (106) in which monocytes/macrophages were removed with anti-CD14-coated magnetic beads (C) or purified macrophages (106) isolated with anti-CD14-coated beads (D) were treated without or with 1 μM S1P, as indicated. Supernatants were collected at indicated times, and IL-6 secretion was determined by ELISA. Similar results were obtained with three (A, B) or two (C, D) additional cord blood cultures.

To determine whether IL-6 was derived from MCs or from monocytes/macrophages that are also present in the hematopoietic precursor cultures, monocytes/macrophages were immunodepleted with anti-CD14-coated magnetic beads, as CD14 is a membrane-associated glycosylphosphatidylinositol-linked protein expressed at the surface of macrophages but not by MCs. When these macrophage-depleted MCs were cultured in the presence of S1P, there was no detectable production of IL-6 (Fig. 3C), suggesting that the monocytes/macrophages are the source of IL-6. Indeed, macrophages isolated from CB cultures, by virtue of their expression of CD14, secreted large amounts of IL-6 in response to S1P (Fig. 3D). Significant IL-6 secretion was evident within 5 h after addition of S1P, reaching maximum levels at around 10 h (Fig. 3D).

It was of interest to examine the fate of S1P added to CB-MCs. The culture medium contains only 1.6 nM S1P, as measured by LC-ESI-MS/MS. Five minutes after the addition of 1 μM S1P to CB-MCs, there was no significant decrease in S1P levels; however, only one-third remained after 1 h (Fig. 4A). Although the concentration of exogenous S1P in the medium decreases rapidly, even after 24 h, the S1P concentration is still significantly elevated (Fig. 4A). Interestingly, treatment of 8-wk CB-MC cultures with 1 μM S1P for 1 or 2 d was sufficient to significantly induce chymase expression (Fig. 4B). In agreement with the observation that S1P induced rapid secretion of IL-6 from CB cultures (Fig. 3B), the presence of neutralizing anti-IL-6 antibody significantly decreased both IL-6- and S1P-induced chymase expression (Fig. 4C), further supporting a role of IL-6 in this process.

Figure 4.

Figure 4

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Time course of S1P disappearance and effects on chymase expression. A) S1P levels in medium before and after addition of S1P (1 μM) to cord blood progenitors cultured for 8 wk with SCF alone were determined at the indicated times by LC-ESI-MS/MS. B, C) Cord blood progenitors cultured for 8 wk with SCF alone were treated without or with 1 μM S1P for the indicated times (B) or without or with 1 μM S1P or 50 ng/ml IL-6 in the absence or presence of anti-IL-6 antibody for 48 h, as indicated (C). Percentage of chymase-expressing MCs was determined as described in Materials and Methods.

S1P triggers degranulation and induces functional features of CB-MC-expressing chymase

Cord blood progenitor cultures produce and secrete CCL2, also known as monocyte chemoattractant protein-1 (MCP-1), which was enhanced by culturing in the presence of S1P (Fig. 5A). Although CB cultures spontaneously secrete CCL2, treatment with S1P induced significant increases compared to CB cultures exposed to SCF alone, in the absence or presence of vehicle (Fig. 5B). Importantly, S1P induced secretion of CCL2 from purified CB-MCs depleted of monocytes/macrophages (Fig. 5C). A significant increase was observed within 2 h after addition of S1P to purified CB-MCs (devoid of monocytes/macrophages) and increased thereafter (Fig. 5C). Levels of CCL2 remained elevated for at least 24 h after the addition of exogenous S1P (Fig. 5C). Because these MCs express S1P1 and S1P2 receptors (19), we next examined which of the receptors was involved in S1P-induced CCL2 secretion. The S1P2 antagonist (30), JTE-013, markedly reduced CCL2 secretion in response to S1P, whereas VPC23019, an antagonist of S1P1(30), had no significant effect (Fig. 5D). In sharp contrast, although macrophages are capable of releasing large amounts of CCL2, no significant stimulation was observed in response to S1P (Supplemental Fig. 1A).

Figure 5.

Figure 5

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S1P enhances CCL2 release from purified CB-MCs but not from purified CB-macrophages. A) Cord blood progenitors were cultured for 8 wk with SCF (100 ng/ml) alone (open bar) or in the presence of S1P (0.1 and 1 μM, solid bars). Cells (106) were then stimulated again with SCF in the absence or presence of S1P for 1 wk. Supernatants were collected, and CCL2 levels were determined by ELISA. B) Cord blood progenitors were cultured for 8 wk with SCF (100 ng/ml) alone (none; circles), or in the presence of vehicle (triangles) or 1 μM S1P (squares). Cells (106) were then stimulated again for the indicated times, and CCL2 in supernatants was measured by ELISA. C–D) After 8 wk in culture with SCF alone, purified CB-MCs (1×106), in which monocytes/macrophages were removed with anti-CD14-coated beads, were treated without or with 1 μM S1P (C) or pretreated for 30 min with vehicle, 1 μM JTE-013, or 1 μM VCP23019 prior to stimulation without or with 1 μM S1P (D). At indicated times (C) or after 24 h (D), supernatants were collected, and CCL2 secretion was determined by ELISA. Similar results were obtained with three (A, B) or two (C, D) additional cord blood cultures. *P < 0.01 vs. S1P treatment.

An important functional difference between double-positive MCTCs and lung-like MCTs is that the former are also known to respond in an IgE-independent manner to a number of secretagogues, such as polyamines like compound 48/80, and the anaphylatoxin C5a (23). To examine the functional characteristics of chymase-expressing CB-MCs developed in the presence of S1P, their capacity to degranulate in response to antigen, C5a, and 48/80 was determined by β-hexosaminidase release. In agreement with previous studies (10), MCTs developed in the presence of SCF alone readily degranulated in response to crosslinking of FcεRI by antigen and substance P but did not respond to C5a or 48/80 (Fig. 6A). However, CB-MCs developed in the presence of S1P, which increases chymase and C5aR expression (Fig. 2), degranulate in response to C5a (Fig. 5A), similar to skin-derived MCTCs (13). Furthermore, these MCs were also degranulated by compound 48/80 (Fig. 6A). As expected, ionomycin and substance P induced similar degranulation in both phenotypes of MCs (Fig. 6A). In addition, antigen, substance P, and ionomycin, but not C5a and 48/80, enhanced secretion of CCL2 from MCTs developed in the presence of SCF alone (Fig. 6B). Conversely, C5a and 48/80 only enhanced release of CCL2 from MCTCs, developed in the presence of S1P (Fig. 6B).

Figure 6.

Figure 6

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CB-MCs generated in the presence of S1P acquire functional features of skin MCTCs. After culturing for 8 wk with SCF in the absence (open bars) or presence of S1P (solid bars), purified CB-MCs were stimulated for 2 h with IgE/Ag, C5a (1 μg/ml), compound 48/80 (1 μg/ml), substance P (1 μM), or ionomycin (1 μM). A) Degranulation was assessed by β-hexosaminidase release. B) CCL2 in supernatants was measured by ELISA. Similar results were obtained with two additional cord blood cultures from other donors. *P < 0.01 vs. vehicle treatment.

DISCUSSION

S1P has been added to the repertoire of mediators produced and released by MCs that, in turn, regulate MC functions (16,17,18,19, 31,32,33,34). FcεRI triggering has been shown to utilize SphK, the enzyme that produces S1P, to mobilize Ca2+ from internal stores, an event necessary for degranulation (31, 33). Moreover, the balance between sphingosine and S1P determined by SphK is decisive for allergic responsiveness of MCs (32). Secreted S1P is able to bind and activate its receptors on MCs. S1P1 induces cytoskeletal rearrangements, leading to the movement of MCs toward an Ag gradient, whereas S1P2 is required for the degranulation response (18, 19). S1P also increased expression of MCP-1, MIP-1α, MIP-1β, and MIP-2 in MCs, all important modulators of monocyte, macrophage, and eosinophil recruitment and inflammation (18, 19, 35). Production of S1P in MCs has grown more complex with the recent demonstration that both SphK1 and SphK2 are activated on FcεRI engagement (16) and are important in vivo for MC-dependent anaphylactic responses in mice (17). These findings together with the observation that SCF, an important growth factor required for MC survival and differentiation, also activates SphK1 and SphK2 (16), emphasize the important role of S1P generation in MC physiology.

Here, we report that S1P also accelerates the generation of MCs from hematopoietic progenitors and strikingly increases chymase expression. These CB- MCTCs have functional FcεRIs and, similar to skin MCTCs, are also activated by the anaphylatoxin C5a and the secretagogue 48/80. Thus, MCTCs are functionally distinguished from MCT phenotypes of human MCs, suggesting important differences that may affect their participation in disease states. The ability of MCTCs to be activated by agents not associated with FcεRI and IgE suggest that this cell type may have a greater role in innate immunity by responding to either innate or microbial danger signals. Although human MCs can be generated from umbilical cord blood progenitors cultured in medium supplemented with SCF and varied accessory factors, including combinations of cytokines (6, 9, 36, 37), remarkably, the percentage of MCs expressing chymase is similar to what we found utilizing SCF with S1P alone. Interestingly, the responses of MCTCs to C5a and 48/80 are much greater than expected. There are several possible explanations for this. First, it is well established that MCs of different maturity also differ in their histamine content and their ability to respond to cell activation (38). Sk-MCTCs release much more histamine in response to IgE/Ag than lung-derived MCTs (39). Indeed, MCTC granules generally are more uniformly electron dense, larger, and more numerous than MCT granules (40). Alternatively, a higher proportion of MCs may be expressing C5aR than chymase, or smaller amounts of C5aR are needed for a functional response. It is also possible that C5a might activate MCTCs to release another factor that can activate MCTs through a pathway other than through C5aR.

Although LPA, a serum-borne lysophospholipid structurally closely related to S1P, has been shown to accelerate MC proliferation and differentiation to tryptase expressing MCTs; interestingly, it had no effect on the small number of chymase-expressing cells nor did it increase chymase activity (20). The ability of S1P to induce expression of chymase and C5aR is most probably mediated via release of IL-6 from cord blood progenitor cultures that contain adherent macrophages. Indeed, highly purified monocytes/macrophages from these cultures released IL-6 in response to S1P. Macrophages express multiple S1P receptors, of which S1P1 and S1P2 predominate (41,42,43). In agreement, inhibiting S1P1 and S1P2 with specific antagonists significantly reduced S1P-stimulated IL-6 release from macrophages (Supplemental Fig. 1B). Of note, S1P stimulated secretion of CCL2 from MCs independent of the presence of macrophages.

Our data suggest crucial roles for S1P in regulating development of hematopoietic progenitors into functionally mature MCs expressing chymase and reveal a complex interplay between macrophages and MCs during the development of fully differentiated MCs (Fig. 7). According to this model, S1P induces secretion of IL-6 from monocytes/macrophages and CCL2 from MCs. IL-6, in turn, may act on progenitors, enhancing the mitogenic and survival effects of SCF and promoting development and maturation of MCs and inducing chymase and C5aR expression (10). In addition, S1P induces CCL2 release from MCs to recruit monocytes/macrophages to their vicinity, thereby enhancing the interaction between these different types of cells. In this regard, an elegant study in mice demonstrated that adult MC progenitors are derived directly from multipotential progenitors instead of, as previously proposed, common myeloid progenitors or granulocyte macrophage progenitors (44). Moreover, these MC-committed progenitors can give rise to both connective tissue-type and mucosal-type MCs, which is determined by factors present in the site of differentiation (44).

Figure 7.

Figure 7

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Proposed model of human MC development and the involvement of S1P. S1P stimulates release of IL-6 by monocytes/macrophages. In turn, IL-6 can act on developing MCs at different stages of development, promoting proliferation and inducing chymase expression. S1P also enhances CCL2 secretion from MCs, which is a chemoattractant for monocytes/macrophages, further enhancing crosstalk between monocytes/macrophages and MCs in response to S1P. For simplicity, a multipotential progenitor capable of developing into MCTs and MCTCs is depicted.

We found that S1P can also induce release of IL-6 from macrophages. Interestingly, previous results have suggested that SphK1, which produces S1P, plays a key role in the generation and release of proinflammatory mediators from human macrophages triggered by anaphylatoxins (45) and in neutropenia, peritonitis, and cytokine production in vivo(46). Anaphylatoxin C5a, one of the complement fragments produced by activation of the complement system, is involved in a variety of disorders in which MCs play critical roles, including septic shock and adult respiratory distress syndrome. Our finding that S1P enhances MC expression of C5aR and their ability to respond to C5a further supports the notion of a potential role of S1P in anaphylatoxin-triggered inflammatory responses in vivo(45, 46).

Chymase, a chymotrypsin-like serine protease that is only secreted from MCTCs, has been associated with sepsis in various mouse models (47,48,49). It has been suggested that increased intracellular chymase activity leads to enhanced microbiocidal activity directly or may function indirectly. Extracellularly, MC chymase can degrade endothelin-1, a potent constrictor of blood vessels that has been implicated in vascular changes associated with sepsis (50) and cleave chemokine precursors to generate activated chemokines that recruit neutrophils to bacterial infections (49). Thus, murine MCs, which express at least four chymase proteins, have the potential to help (48, 49), rather than harm. Nonetheless, earlier studies found that mice with MCs deficient in chymases usually survive peritonitis induced by cecal ligation and puncture better than wild-type mice (47). The serine peptidases seem to increase mortality by cleaving survival-enhancing cytokines, such as IL-6 (47). Similarly, it has been demonstrated that a variety of cytokines produced by cultured human skin MCTCs, including IL-5, IL-6, IL-13, and TNF-α, are cleaved by MC peptidases, primarily chymase (29). However, there is only a single chymase gene in humans (51). Recently, it was demonstrated that lipopolysaccharide up-regulates chymase expression in human MCs, suggesting that a gram-negative bacterial infection may induce MCs to express a unique composition of proteases beneficial for controlling and eliminating the infection (52). Although chymase expression has been reported to be elevated in individuals dying from anaphylaxis (53), its functions in sepsis and anaphylaxis are still not well understood (51, 54). Our results demonstrate an important role for S1P in regulating development of functionally mature chymase expressing human MCs and their functions.

Supplementary Material

Supplemental Data
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Acknowledgments

We thank the Labor and Delivery team at the VCU Health System for kindly providing us with human umbilical cord blood samples and Amgen (Thousand Oaks, CA, USA) for stem cell factor. We also acknowledge the helpful discussions with Drs. Lawrence Schwartz, Daniel Conrad, and John Ryan. This work was supported by National Institutes of Health grants RO1AI500941 and U19AI077435-018690 (S.S.) and K01AR053186 (C.A.O.), and the Intramural Research Program of the National Institute of Mental Health (S.M.).

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