Published online before print October 11, 2006
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* Bioxell SpA, Milano, Italy; and
Laboratory of Experimental Medicine and Endocrinology, K. Universiteit of Leuven, Leuven, Belgium
1Correspondence: BioXell S.P.A., Via Olgettina, 58, 20132 Milano, Italy. E-mail: enrico.baroni{at}bioxell.com
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RI, and IL-3 receptor-
chain, and depends on the expression of the vitamin D receptor (VDR). It is important that mast cell progenitors obtained from VDR-ablated mice undergo an accelerated maturation in vitro and give rise to more responsive mast cells than wild-type. Furthermore, histological analysis of mast cell density in peripheral tissues reveals a moderate increase in the number of mast cells in the skin of VDR-deficient mice compared with wild-type animals. These data support the hypothesis of a physiological role of 1,25(OH)2D3 in mast cell development and suggest novel, therapeutic uses of 1,25(OH)2D3 analogs.
Key Words: apoptosis • maturation • bone marrow • cell surface molecules
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The immunoregulatory properties of VDR ligands have been studied in different models of autoimmune diseases and allograft rejection. However, the cellular and molecular targets for anti-inflammatory activity of 1,25(OH)2D3 appear to be complex and remain not fully elucidated [4 , 5 ].
Mast cells (MCs), which act as potent triggers and amplifiers of inflammatory and allergic responses, are one of the main effector cells of the Th2 response. Cross-linking of the high-affinity FcRI by multivalent antigens bound to IgE antibodies provides the main immunological stimulus for MC activation, operating in type I hypersensitivity and anaphylactic reactions [6
7
8
]. MCs are also triggered by a variety of immunological and nonimmunological stimuli, such as complement components, cytokines, bacterial and viral constituents, kinins, and neuropeptides [9
, 10
]. The activation of MCs not only causes the release of preformed, granule-associated mediators but also initiates the de novo synthesis of lipid-derived substances and cytokines [11
].
MCs are the progenies of hematopoietic stem cells. Progenitor cells leave the bone marrow and after entering the circulation as undifferentiated cells, seed peripheral tissues such as lung, bowel, and skin, where they finally differentiate into mature MCs in response to yet poorly defined signals. Mouse MCs (mMCs) can be generated in large amounts from bone marrow cells cultured in the presence of IL-3 [12 ]. The c-kit ligand stem cell factor (SCF) also plays a critical role in MC survival, proliferation, activation, and mediator content release [13 14 15 ].
MCs, like many immune cell types, express VDR and respond to 1,25(OH)2D3 [16
]. Previous studies exploring the effects of 1,25(OH)2D3 on activation of mature MCs reported contrasting evidence, and 1,25(OH)2D3 was reported as an inhibitor of calcium ionophore-induced histamine release by peritoneal MCs [17
] and as an enhancer of IgE-mediated C57 MC line degranulation [18
]. However, the biological effects of 1,25(OH)2D3 on MCs remain largely unexplored. In this paper, we describe extensive studies aimed at characterizing the biological activities of 1,25(OH)2D3 on MC proliferation, survival, differentiation, and function. Our results suggest that 1,25(OH)2D3, although only partially affecting release of inflammatory mediators upon FcRI cross-linking, inhibits MC differentiation by promoting apoptosis of MC precursors. Furthermore, the lack of VDR signaling in VDR-deficient bone marrow cells leads to an accelerated differentiation into mature MCs in vitro. Moreover, histological analysis of MC density in different tissues reveals a moderate increase in the number of MCs in the skin of VDR-deficient mice compared with wild-type animals, which does not relate to changes in levels of serum calcium. These observations, beside demonstrating a negative effect of 1,25(OH)2D3 on MC precursors, support a role for VDR in MC development under physiological conditions.
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Animals
BALB/c mice (8- to 12-weeks old, female, 18–22 g), purchased from Charles River Laboratories (Calco, Lecco, Italy), were used in all experiments unless otherwise specified. Swiss mice-derived VDR-knockout (Leuven VDR KO) mice and relative control littermates [19
] were used in the analysis after genotyping by PCR analysis of genomic DNA extracted from tail biopsies. They were fed on a normal diet or on a calcium-enriched diet to prevent hypocalcemia as a result of the absence of VDR activity. All mice were bred in the animal housing facilities of K. Universiteit Leuven (Proefdierencentrum Leuven, Belgium). The Institutional Review Board has approved all animal studies.
Isolation and culture of mouse bone marrow cells
Femurs from BALB/c or Leuven female mice were cleaned and cut at their edge. Bone cavity was washed three times with RPMI 1640 supplemented with 15% FCS (HyClone, Logan, UT), glutamax (Gibco-BRL, UK), pen/strep (Gibco-BRL), and 50 µM ß-ME. Cell suspension was filtered through 70 µm pore membrane (352350, BD Falcon) and then, washed once with complete medium. Prior to culture at 37°C with 5% CO2, 0.5% of an IL-3-containing supernatant, prepared from the transgenic mouse cell line topoisomerase 3-IL-3, was added to the cell suspension. When required, recombinant (r)SCF (455-MC/CF, R&D Systems, Minneapolis, MN) was added at the concentration of 100 ng/ml. Cells were kept at a concentration ranging from 5 x 105 to 1 x 106 cells/ml, changing medium and flask every 4–7 days. In some cases, cultures were enriched in hematopoietic progenitors using a lineage cell depletion kit (130-090-858, Miltenyi Biotec, Auburn, CA).
RT-PCR
Total RNA was extracted from cells using a commercial kit (74104, Qiagen RNeasy mini kit, Qiagen, Valencia, CA), followed by treatment with DNase I. RNA concentrations were finally quantified with a Nanodrop® ND-1000 spectrophotometer.
RT was performed with RT reagent (N-8080234, Applied Biosystems, Foster City, CA) with random hexamers (according to the manufacturer’s instructions). Real-Time PCR was performed in 96-well optical reaction plates (Applied Biosystems). The amplification master mix was prepared according to the following protocol (volumes refer to a single well with a final volume of 40 µl/well): 2x TaqMan® Universal PCR Master mix (4304437, Applied Biosystems), 20 µl; 20x assay target gene, 2 µl; H2O, 8 µl; cDNA, 10 µl. Reactions were run on a SDS 7000 (Applied Biosystems) instruments with the following amplification program: 2 min at 50°C; 10 min at 95°C; 15 s at 95°C; and 1 min at 60°C for 40 cycles. Cycle threshold (Ct) values were then exported, and relative quantitations were performed using the 
Ct method. All primers/probe assays used were purchased from Applied Biosystems and carried the FAM reporter. Assays used include hypoxanthine guanine phosphoribosyl transferase (HPRT; Mm00446968_m1), VDR (Mm00437297_m1), and Cyp24 (assay-by-design, mCYP24-EX56, forward primer: 5'-ACAGCGAGCTGAACAAATGGT-3', reverse primer: 5'-AGGAGCCCGAATCTCTTCTCATAT-3', reporter sequence: 5'-CAAGGCAGATGCTTTCA-3').
Cell proliferation assay
Freshly isolated bone marrow cells were cultured in Costar 96-well plates, round bottom (1.5x104 cells/well), with or without murine rIL-3 (0.5%), in the presence or absence of increasing concentrations of 1,25(OH)2D3 (1, 10, 100, 1000 nM), with or without 100 ng/ml SCF. After 8 days of culture, plates were centrifuged and after removal of the supernatant, frozen at –80°C to lyse the cells. CyQUANT cell proliferation assay kit (Molecular Probes, Junction City, OR) was used for measuring cell proliferation, according to the manufacturer’s instructions.
FACS analysis
A Becton Dickinson (San Jose, CA) LSR instrument was used for all FACS analyses. Cell sorting was performed at the core facility of San Raffaele Hospital (HSR, Milan, Italy) using a Becton Dickinson FACSVantage SE with FACSDiva option.
Stainings with annexin V and propidium iodide (PI) were performed with the annexin V apoptosis detection kit I (BD PharMingen, San Jose, CA, Cat. No. 556547) to determine the percentage of apoptotic and dead cells in the cultures. Only cells appearing as single-positive staining for annnexin V were considered apoptotic, and those appearing as double-positive staining for annexin V and PI were gated as dead cells.
The following antibodies were used to check cell differentiation: PE-anti c-kit (PE anti-CD117, 553869, BD PharMingen), FITC anti-IgE (553415, BD PharMingen), purified IgE (IgE anti-TNP, 557079, BD PharMingen), PE anti-IL-3 receptor- chain (IL-3R; CBL 1362P, Chemicon, El Segundo, CA), FITC anti-c-kit (553354, BD PharMingen).
Cell stimulations and assays for inflammatory mediators release
Mature bone marrow-derived MCs (BMMCs) were stimulated by plating onto 96-well plates previously coated with purified anti-TNP IgE antibodies (557079, BD PharMingen). After 30 min of stimulation, ß-hexosaminidase levels were measured in the culture supernatants and in control cell lysates by hydrolysis of p-nitrophenyl-N-acetyl-ß-D-glucopyranoside (Sigma-Aldrich, St. Louis, MO) in 0.1 M sodium citrate buffer (pH 4.5) for 40 min at 37°C. The percentage of ß-hexosaminidase release was calculated as described previously [20
].
To determine levels of other inflammatory mediators, supernatants were collected after 24 h of stimulation with plate-bound IgE antibodies and stored at –80°C until quantitation. Upon analysis, samples were thawed and assayed for the content of IL-6 and IL-13 (R&D Systems mini kit), leukotriene C4 (LTC-4; 520211, Cayman LTC-4 enzyme immunoassay kit, Cayman Chemicals, Ann Arbor, MI), and mMC-derived protease 1 (mMCP-1; MMCP-1 ELISA kit, Moredun Scientific Ltd., UK).
Assays were performed on at least three replicates and repeated at least three times.
Cytospin preparations
Cells were centrifuged (Shandon Cytospin 4) at 1500 rpm for 5 min, using Shandon Single Cytofunnel (5991040, Shandon, Thermo Electron Corp., Waltham, MA) sample chambers on Menzel-Glaser (SuperFrost Plus, J1800AMNZ) glass slides, which were then stained with toluidine blue (Bio-Optica, Ireland) or with anti-VDR antibody (MA1-710, Affinity BioReagents, Golden, CO) for evaluation of cell phenotype by microscopic examination. Images were taken with a Leica DC200 camera mounted on a Leica HC microscope.
Histological preparations and samples evaluation
Upon sacrifice, skin samples were embedded in OCT (BDH, Milan, Italy) and frozen in liquid nitrogen-cooled isopentane. Frozen tissues were then cut (5 µm thickness) and stained with toluidine blue (Bio-Optica) for MC density evaluation. Number of cells over 10 high-powered fields (HPF; 1 HPF=2.3 mm2) was determined. Results are reported as number of MCs per mm2 dermis.
Measurement of serum calcium levels
Upon sacrifice, blood samples were collected from wild-type and VDR-KO animals. Briefly, sera were prepared by incubation at 37°C followed by centrifugation at 4°C and removal of precipitates. Calcemia was evaluated by using a commercial colorimetric kit (Calcium Dry-Fast, Sentinel, Milan, Italy).
Statistical analyses
Student’s t-test analysis was performed when comparing two groups, and one-way ANOVA followed by Dunnet’s post-hoc analysis was performed to compare more than two groups.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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RI surface expression, which can be detected easily by staining with IgE antibodies followed by staining with anti-IgE antibodies [21
]. Efficiency and specificity of the staining method were confirmed by parallel analysis of FcRI mRNA expression (data not shown). After 5 weeks in culture, we obtained a population, mainly consisting of mature BMMCs, as defined by their double-positive staining for c-kit and FcRI receptors (data not shown) and by morphological analysis (Fig. 1A
). Furthermore, staining the same cells with anti-VDR antibody revealed expression of the VDR, mostly localized in the nucleus (Fig. 1B)
.
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Figure 1. VDR expression and responsiveness to 1,25(OH)2D3 of mouse BMMCs. (A) Toluidine blue staining of mature BMMCs. (B) Staining of mature BMMCs with anti-VDR antibody. (C) Quantitative RT-PCR analysis of mature BMMCs. Cells were cultured for 24 h in the presence or absence of 100 nM 1,25(OH)2D3 before being collected and analyzed for gene expression. The y-axis reports the values of expression relative to the housekeeping gene (HPRT). P < 0.05 for all comparisons.
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1,25(OH)2D3 reduces release of inflammatory mediators upon activation of mature BMMCs
The potential effects of 1,25(OH)2D3 on activation of mature MCs were then investigated. It is well known that large amounts of inflammatory mediator release, including ß-hexosaminidase and LTC-4, and expression of several inflammatory genes are induced following FcRI cross-linking on fully mature BMMCs [23
24
25
].
To evaluate if 1,25(OH)2D3 could affect degranulation of mature BMMC, we incubated fully mature MCs with 1,25(OH)2D3 for 24 or 48 h, before plating them into 96-well plates previously coated with IgE antibodies. After 30 min, supernatants were collected and assayed for ß-hexosaminidase activity. Although 24 h preincubation with 1 µM 1,25(OH)2D3 did not affect ß-hexosaminidase release in the assay buffer, 48 h treatment significantly reduced exocytosis induced by the two highest concentrations of IgE antibodies (Fig. 2A ). Release of IL-13, mMCP-1 and LTC-4 was also assayed, after 24 h stimulation following 96 h preincubation with 1 µM 1,25(OH)2D3. As shown in Figure 2B , treatment with 1 µM 1,25(OH)2D3 moderately, but significantly, reduced release of mMCP-1 and LTC-4, and release of the cytokine IL-13 was unchanged. Viability of mature BMMCs was not affected by 1,25(OH)2D3 treatment, neither in these assays nor after longer incubation times (data not shown).
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Figure 2. 1,25(OH)2D3 negatively regulates release of inflammatory mediators by BMMCs after cross-linking of FcRI. BMMCs obtained from wild-type or VDR-KO mice were incubated in the presence or absence of 1,25(OH)2D3 and stimulated by plating onto 96-well plates previously coated with purified IgE antibodies. (A) Following 24 or 48 h of exposure to 1 µM 1,25(OH)2D3 and after 30 min of stimulation, supernatants were collected and assayed for the levels of ß-hexosaminidase; results are expressed as percent of total cell content of ß-hexosaminidase, as revealed by assay of equal number of cells lysed with Triton X-100. (B) Following 96 h of exposure to 1 µM 1,25(OH)2D3 and after 24 h of stimulation, supernatants were assayed for the content of IL-13, mMCP-1, and LTC-4. All experiment were repeated at least three times. *, P < 0.05; **, P < 0.01. ns, Not significant.
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Expression of VDR and responsiveness to 1,25(OH)2D3 in BMMCs
We then asked if 1,25(OH)2D3 could exert stronger effects on BMMC precursor proliferation and differentiation rather than on activation of mature MCs. First, to determine the responsiveness of mouse bone marrow cells to 1,25(OH)2D3, we incubated freshly isolated bone marrow cells in the presence or absence of 1,25(OH)2D3 and purified RNA to analyze expression of VDR and Cyp24. Real-time PCR analysis confirmed expression of VDR in bone marrow cells and revealed a strong induction of Cyp24 gene expression in the presence of 1,25(OH)2D3 (Fig. 3A
). It is interesting that IL-3 culture of bone marrow cells resulted in induction of VDR expression (Fig. 3A
, left panel).
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Figure 3. VDR expression and responsiveness to 1,25(OH)2D3 of mouse BMMCs. (A) Quantitative RT-PCR analysis of freshly isolated or cultured BMMC. Cells were cultured for 2 days in the presence or absence of 100 nM 1,25(OH)2D3, before being collected and analyzed for gene expression. P < 0.05 for all comparisons. (B) Cell proliferation assay. Freshly isolated bone marrow cells were cultured in IL-3-containing medium, in the presence or absence of 100 nM 1,25(OH)2D3, with or without 100 ng/ml SCF. After 8 days of culture, cell pellets were frozen and analyzed by a commercial cell proliferation assay, which has been performed more than three times. ***, P < 0.001, versus [IL-3+SCF; no 1,25(OH)2D3]; °°°, P < 0.001, versus [IL-3; no 1,25(OH)2D3].
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Dose-dependent induction of apoptosis of BMMC precursors by 1,25(OH)2D3
To determine whether antiproliferative effects of 1,25(OH)2D3 on MC precursors were dependent on induction of cell death, we cultured bone marrow-derived cells in the presence or absence of 1,25(OH)2D3 and analyzed the number of alive and dead cells accumulating during cell culture. We observed that exposure to 1,25(OH)2D3 progressively increased the number of dead cells in the culture, eventually leading to an inability to propagate the culture (data not shown). To assess whether this effect was a result of the induction of apoptosis after a 7-day exposure to 1,25(OH)2D3, we analyzed cell phenotype by staining with annexin V, which binds phosphatidylserine residues exposed on membranes of early apoptotic and dead cells and PI, which stains dead cell genomic DNA. As shown in Figure 4A
, incubation with 1,25(OH)2D3 resulted in a dose-dependent induction of apoptosis of precursor cells, with a maximal proapoptotic effect of similar intensity to that observed upon IL-3 deprivation [26
]. To evaluate the specificity of the observed apoptotic effect on MC precursors, similar experiments were carried out by culturing bone marrow-derived cells from VDR-KO mice and their control littermates. As depicted in Figure 4B
, the induction of apoptosis was detectable in cell cultures derived from VDR-wild-type mice upon exposure to 1,25(OH)2D3. Conversely, no evidence of increased apoptosis was observed when VDR-deficient bone marrow cells were cultured in the same conditions, thus confirming the requirement for VDR expression for the proapoptotic effect of 1,25(OH)2D3. The differences observed in relative percentage of apoptotic cells between Figure 4A
and 4B
, are probably to be attributed to the different genetic background of the mouse strains used (BALB/c mice vs. Swiss mice) [19
].
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Figure 4. 1,25(OH)2D3 triggers apoptosis of mouse BMMC precursors. (A) Freshly isolated bone marrow cells were incubated at 37°C in IL-3-containing medium, with or without increasing concentrations of 1,25(OH)2D3. After 8 days, cells were harvested and analyzed by flow cytometry after staining with FITC-annexin V and PI to determine the percentage of apoptotic and dead cells in the cultures. (B) Bone marrow cells derived from wild-type (wt) or VDR-KO mice and cultured with or without 100 nM 1,25(OH)2D3 were analyzed after 8 days of culture. Three independent cell cultures were derived from different animals with similar results. One representative of the three cultures is shown. Cells appearing as single-positive staining for annexin V are considered early apoptotic, and those appearing as double-positive staining for annexin V and PI are considered dead cells. These experiments have been repeated more than four times with similar results.
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RI cell surface expression. In the presence of IL-3, bone marrow-derived cells first acquired expression of c-kit receptor or FcRI alone and subsequently became a double-positive FcRI+c-kit+ (data not shown). As FcRI+c-kit– cells arised early and transiently in the culture, we asked if FcRI+c-kit– cells represented a precursor of mature FcRI+c-kit+ MCs. We therefore purified FcRI+c-kit– (Fig. 5
, lower panels) and FcRI+c-kit+ (Fig. 5
, upper panels) populations from a 10-day-old bone marrow cell culture and cultured the purified cells in IL-3-containing medium for several days to monitor their differentiation. As shown in Figure 5
, after 24 h of culture, 16% of FcRI+c-kit– cells had already acquired a FcRI+c-kit+ mature MC phenotype, and this percentage increased to 87% in the subsequent 8 days of culture. Morphological analysis of these newly generated double-positive cells revealed that they were truly mature MCs, as defined by their characteristic granulated morphology (data not shown).
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Figure 5. Identification of MC precursor stages in mouse BMMC cultures. Bone marrow cells cultured for 10 days in IL-3-containing medium were stained with IgE + FITC-anti-IgE and PE-anti-ckit antibodies and then sorted in the gates as shown. Sorted cells were then recultured in IL-3-containing medium and analyzed at different time-points after staining with the same antibodies. This experiment has been repeated three times.
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RI+c-kit– MC precursors. To answer this question, we cultured freshly isolated bone marrow cells in IL-3-containing medium in the presence or absence of 100 nM 1,25(OH)2D3 and followed their differentiation by FACS analysis for expression of FcRI and c-kit cell surface markers. As shown in Figure 6
, freshly isolated bone marrow cells contained no or few double-positive FcRI+c-kit+ MCs (0.1±0.0% on a total of 4x107 cells, n=6), giving rise in 5 weeks of culture in the presence of IL-3 to a population highly enriched in mature, double-positive MCs (86.0±2.8% on a total of 1.1±0.2x108 cells, n=6). Starting from the sixth day of exposure to 1,25(OH)2D3, we observed a relatively larger decrease of FcRI+c-kit– and FcRI–c-kit+ cell subpopulations among live cells compared with the double-positive population (Fig. 6A)
. Similar results were obtained in several time-course experiments, and in all cases, addition of 1,25(OH)2D3 eventually led to an inability to propagate the cell culture. To ensure continuous exposure to 1,25(OH)2D3, the experimental protocol included the addition of fresh 1,25(OH)2D3 to the culture twice a week. This procedure allowed the maintenance of constantly elevated mRNA levels of the VDR activation marker Cyp24, as determined by RT-PCR analysis at different time-points (data not shown). The relative increase in the FcRI+c-kit+ cell population revealed by FACS analysis was likely to reflect the lower sensitivity of mature MCs versus their precursors to a 1,25(OH)2D3 proapoptotic effect. Accordingly, parallel analysis of cell counts in the cultures revealed that the absolute number of mature MCs was not changing after Day 6, when 1,25(OH)2D3 antiproliferative effects first became apparent (Fig. 6B
, solid bars).
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Figure 6. Proapoptotic activity of 1,25(OH)2D3 targets a FcRI+ precursor of MCs. (A) Bone marrow cells cultured from Day 0, with or without 100 nM 1,25(OH)2D3, were stained at different time-points with IgE + FITC-anti-IgE and PE-anti-ckit antibodies and analyzed by FACS. Upper plots are representative of cells cultured in IL-3, and the lower plots represent cells cultured in IL-3 plus 100 nM 1,25(OH)2D3. (B) The same bone marrow cell cultures were stained with Trypan blue at the indicated time-points, and live cells were counted under optical microscope. Solid bars represent the number of mature MCs, calculated by FACS analysis of c-kit/FcRI staining depicted in A, and open bars represent the number of the remaining live cells. Time-course experiments have been repeated more than six times with similar results.
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RI, c-kit, and IL-3R chain antibodies and analyzed expression of these markers by FACS.
As depicted in Figure 7
, bone marrow cells cultured in the presence of 1,25(OH)2D3 showed a fourfold reduction in IL-3R+FcRI+ cell frequency, as well as a twofold reduction in the frequency of the IL-3R+c-kit+ cell population (Fig. 7A)
when compared with cells cultured in the presence of vehicle (EtOH) alone (Fig. 7B)
. These findings are suggestive of a marked effect of 1,25(OH)2D3 on IL-3R-expressing cells. However, this result was not a result of down-regulation of IL-3R by 1,25(OH)2D3, as no significant down-regulation of IL-3R mRNA and protein levels was detectable before 1,25(OH)2D3 could exert its proapoptotic effect (data not shown). In further support of this, 1,25(OH)2D3 did not affect IL-3R chain levels nor induce apoptosis in the IL-3-dependent cell line BAF-3 (data not shown).
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Figure 7. Proapoptotic activity of 1,25(OH)2D3 targets an IL-3R+ precursor of MCs. Bone marrow cells cultured for 11 days with or without 100 nM 1,25(OH)2D3 were stained with PE-anti-IL-3R chain and IgE + FITC-anti-IgE (A) or FITC-anti-CD117 (B) antibodies and analyzed by FACS. One selected experiment is shown, representative of several others performed with similar results. This experiment has been repeated three times with similar results.
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RI+c-kit– and FcRI–c-kit+ cell populations from a 15-day-old bone marrow cell culture and then cultured each purified population in the presence or absence of 100 nM 1,25(OH)2D3 for several days. FACS analysis revealed that although 1,25(OH)2D3 inhibited transition of FcRI+c-kit– cells into mature, double-positive MCs (Fig. 8A
), it was not triggering apoptosis of FcRI+c-kit– cells, as indicated by morphological analysis by FACS (Fig. 8B)
. Reliability of SSC/FSC analysis for evaluation of cell morphology to assess cell viability qualitatively and quantitatively was confirmed by analysis of parallel cell culture stainings with annexin V/PI and FACS analysis (data not shown).
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Figure 8. Inhibition of MC precursor maturation by 1,25(OH)2D3. FcRI+-c-kit– (A, B) or FcRI–-c-kit+ (C, D) cells were sorted from a 15-day-old BMMC culture in IL-3 after staining with IgE + FITC-anti-IgE and PE-anti-ckit antibodies. Purified cells were then recultured in IL-3-containing medium in the presence or absence of 100 nM 1,25(OH)2D3 and analyzed at different time-points after staining with the same antibodies (A, C). For the last time-point, cell morphology is reported as analysis of forward-scattering (FSC) and side-scattering (SSC) for FcRI+-c-kit– (B) or FcRI–-c-kit+ (D) cell-derived cultures. This experiment has been repeated three times with similar results.
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RI–c-kit+ cells were cultured in the presence of IL-3, a transient loss of expression of c-kit receptor occurred, followed by the acquisition of FcRI expression, prior to the final maturation to double-positive FcRI+c-kit+ MCs. Addition of 1,25(OH)2D3 to the culture of FcRI–c-kit+ cells resulted in reduced percentage of FcRI+c-kit+ cells (Fig. 8C)
and induction of cell death, as evident by analysis of cell morphology (Fig. 8D)
. It is interesting that 1,25(OH)2D3 appeared to exert its proapoptotic effect concomitantly with the acquisition of FcRI expression on the cell surface (data not shown).
VDR expression is required for 1,25(OH)2D3-mediated modulation of MC precursor differentiation
To confirm the specificity of the effects of 1,25(OH)2D3 on BMMC cultures, we analyzed the phenotype of VDR-KO mice- and control littermate-derived bone marrow cells cultured in the presence of 1,25(OH)2D3. As depicted in Figure 9A
, VDR-KO bone marrow cells were able to generate mature MCs, regardless the addition of 1,25(OH)2D3, whereas development of MCs from wild-type mice was inhibited by 1,25(OH)2D3.
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Figure 9. MC development in the absence of VDR signaling in vitro. (A) Ten-day-old bone marrow cells derived from wild-type or VDR-KO mice and cultured with or without 100 nM 1,25(OH)2D3 were stained with IgE + FITC-anti-IgE and PE-anti-CD117 antibodies and analyzed by FACS. Each graph is representative of three independent cultures derived from different animals. (B) Bone marrow cells derived from wild-type or VDR-KO mice, bred on normal (N) or a calcium-supplemented (Ca) diet were cultured in IL-3-containing medium and analyzed by FACS at the indicated time-points after staining with IgE + FITC-anti-IgE and PE-anti-CD117 antibodies. This experiment has been repeated three times with similar results.
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Lack of VDR expression leads to generation of more responsive MCs in vitro and to higher densities of mature MCs in the skin
Finally, we have asked if VDR basal activity might play a role in determining mature MC activity. We therefore studied activation of in vitro-differentiated MCs obtained from VDR-KO mice and control littermates. Although no difference was observed on IgE-induced degranulation between wild-type and VDR-KO MCs (data not shown), mature VDR-KO MCs were found to be more responsive than wild-type cells to FcRI receptor cross-linking stimulation in terms of secretion of IL-6, IL-13, or LTC-4 (Fig. 10A
). We obtained similar results with cells obtained from animals that were fed with a normal diet or with a diet containing high levels of calcium, thus proving that the differences observed were not a result of alterations in blood calcium levels. Indeed, analysis of calcium levels in sera revealed that although lack of VDR per se resulted in the expected decrease in calcium levels, feeding these animals with a calcium-enriched diet induced a marked raise in calcium levels compared with wild-type control littermates (Fig. 10B)
.
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Figure 10. In vivo development and MC responsiveness in the absence of VDR signaling. (A) BMMCs obtained from wild-type or VDR-KO mice were stimulated by plating onto 96-well plates previously coated with purified IgE antibodies, and activation was assayed by measuring secretion of IL-6, IL-13, and LTC-4 in the culture supernatant. *, P < 0.05 vs. IgE-stimulated wild-type; ***, P < 0.004 vs. IgE-stimulated wild-type. (B) Calcium levels were evaluated in sera prepared from wild-type and VDR-KO animals upon sacrifice. *, P < 0.05, versus wild-type; **, P < 0.01, versus wild-type. (C) Skin tissue samples obtained from wild-type or VDR-KO mice were stained with toluidine blue, and mature, granulated MCs were counted. Results are reported as number of MCs/mm2 dermis. *, P < 0.05, versus wild-type.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MCs act as potent effectors of innate as well as adaptive immune responses, and their activity requires strict regulation. Indeed, disregulated MC activity is associated with severe inflammatory diseases such as allergy and asthma, inflammatory bowel disease, and multiple sclerosis [6 , 29 30 31 ]. A common feature of these disorders is represented by an increased number of mature MCs in the inflamed tissues. This is also observed in systemic mastocytosis, a clonal neoplasm of the MC hematopoietic progenitor associated with gain-of-function mutations involving the tyrosine kinase domain of c-kit [30 , 32 ]. Several efforts have been made in the past years to identify drug candidates able to treat these pathologies by inhibiting MC activation [33 34 35 ]. Preventing MC precursor differentiation into mature MCs may represent an alternative approach to treat MC-dependent diseases, aimed at reducing the number of responsive MCs in the tissues.
mMCs can be generated in large amounts from bone marrow cells cultured in the presence of IL-3 [12
]. Numerous other growth factors, including IL-4, IL-9, IL-10, nerve growth factor, and SCF, have been shown to be important for MC development [36
, 37
]. In particular, SCF, a hematopoietic cytokine that exerts its biological functions by virtue of binding to the c-kit receptor, plays, together with IL-3, a critical role in MC survival, proliferation, activation, and mediator content release [13
14
15
]. Using IL-3-deficient mice, it has been shown that IL-3, although not essential for the generation of MC under physiological conditions, contributes to increased numbers of tissue MCs and immunity to nematode infections [38
]. Moreover, IL-3 perfusion in W/Wv mice, which almost completely lack cutaneous MCs, restores a normal number of MCs in the skin [39
], confirming the important role of IL-3 in MC development. It has been proposed recently that the stage of the mouse BMMC-committed progenitor (MCP) might be identified by a Lyn–Sca-1–Lyc6– CD27–ß7+T1/ST2+c-kit+FcRI– pattern of surface expression [40
, 41
]. According to this, transfer of this cell population into the c-kit-mutant, MC-deficient mice reconstitutes the MC compartment in vivo [41
]. However, the identification of this MCP population does not rule out the possibility that other cell populations develop in vitro or in vivo into MCs or that different intermediate populations appear during the development process.
The findings we show here demonstrate that the FcRI+c-kit– stage might represent a transient MC precursor population. This observation appears to be in contrast with previous studies, suggesting that c-kit expression precedes that of FcRI during in vitro MC development [42
]. Conversely, our findings are supported by earlier evidence describing the presence of a nonbasophil, FcRI+c-kit– MC population in murine bone marrow cultures, which contains fewer granules than mature c-kit-expressing MCs and shows FcRI expression before that of c-kit [43
]. Also, previous studies reported the early expression of FcRI in mouse bone marrow cell culture, which did not correlate with the cellular granule content [44
], and culture of FcRI+ cells sorted from a murine bone marrow cell culture on Day 3 was found to give rise to pure MC populations [45
]. Our study shows that sorted FcRI+c-kit– cells cultured in the presence of IL-3 underwent a differentiation process in the presence of minimal cell proliferation into a cell population composed of 90% FcRI+c-kit+ double-positive, granulated, mature MCs. Furthermore, as depicted in Figure 8C
, when sorted FcRI–c-kit+ cells were cultured in the same way, a surprising, transient loss of expression of the c-kit receptor occurred, followed by the acquisition of FcRI expression alone before final maturation into double-positive FcRI+c-kit+ MCs. One possibility might be that c-kit expression could be transiently modulated in response to endogenously produced SCF or in response to other stimuli or conditions present in the culture.
The aim of this study was to investigate if and how proliferation, differentiation, and survival of MC precursors might be affected by 1,25(OH)2D3. The biological actions of 1,25(OH)2D3 go far beyond the regulation of bone metabolism and calcium homeostasis, including regulation of immunity, angiogenesis and growth, differentiation, and apoptosis of many cell types [1
2
3
]. Most of the studies investigating activity of 1,25(OH)2D3 in regulation of inflammatory response were aimed at demonstrating that 1,25(OH)2D3 exerts inhibitory effects on Th1-type immune responses [5
, 46
, 47
]. However, recent evidences suggested an inhibitory role of 1,25(OH)2D3 also on Th2 responses. In fact, 1,25(OH)2D3 has been shown to inhibit the Th2 cytokine IL-4 in naive T cells during their in vitro polarization [48
], and VDR-deficient mice were found to undergo enhanced IL-4 production by CD4+ lymphocytes and enhanced T cell proliferation in response to IL-4 [49
]. Furthermore, in a study by Wittke et al. [50
], VDR-KO mice were shown to exhibit highly increased levels of IgE under physiological conditions. In their work, the authors showed that VDR deficiency led to an inability to develop experimental allergic asthma, thereby leading to hypothesize an important role for the 1,25(OH)2D3 endocrine system in the generation of Th2-driven inflammation in the lung [50
]. However, in our opinion, such hypothesis is not the sole explanation of the available data. In fact, the extremely high levels of IgE antibodies circulating in VDR-KO mice might saturate MC FcRI receptors, thereby preventing binding of OVA-specific IgE antibodies and MC activation, and in contrast to the suggestion made by Wittke et al. [50
], the high levels of IgE actually indicate a suppressive effect of 1,25(OH)2D3 on the Th2 response. Therefore, those data might not really demonstrate a role for VDR in the generation of Th2-driven inflammation [51
, 52
]. In support of a suppressive effect of 1,25(OH)2D3 on Th2 responses, Topilski et al. [53
] have shown that 1,25(OH)2D3 has anti-inflammatory effects in a Th2-dependent asthma model in vivo, and we have recently described that BXL-628, a synthetic analog of 1,25(OH)2D3, inhibits bladder inflammation in a Th2-dependent, allergic model of interstitial cystitis [54
]. BXL-628 effects included down-regulation of mature MC density within the bladder, as revealed by histological analysis of bladder specimens and by gene expression analysis of several MC markers (mMCP-2, mMCP-4). In this model, in vivo inhibitory activity of the 1,25(OH)2D3 analog BXL-628 was also testified by down-regulation of blood levels of the mMCP-1, which were elevated concomitantly with the onset of inflammation in positive control animals [54
].
The data we showed in the current study provide a possible mechanism of action for 1,25(OH)2D3 in inhibiting MC activity, by means of down-regulating MC development, differentiation, and eventually function. We found that 1,25(OH)2D3 partially inhibits the release of inflammatory mediators by BMMCs stimulated by FcRI cross-linking. This appears to be in contrast with previous studies documenting 1,25(OH)2D3 activity in enhancing IgE-mediated degranulation of the C57 MC line [18
] and supporting earlier evidence of 1,25(OH)2D3 inhibiting calcium ionophore-induced histamine release by peritoneal MCs [17
]. However, the effects on degranulation were partial and only observed at high doses of 1,25(OH)2D3 and therefore, appear to limit the possibility that VDR plays a major role in mature BMMC activation. In contrast, 1,25(OH)2D3 dose-dependently and dramatically prevents the appearance of FcRI+c-kit– cells by inducing apoptosis of earlier progenitor cells and inhibits further maturation of cells that have already acquired the FcRI. These effects are highly specific, as they depend on expression of VDR and provide a basis for further exploring the possibility to use 1,25(OH)2D3 and its analogs in the treatment of MC-related disorders. Moreover, our data suggest that VDR activity might play a role under physiological conditions in regulating MC differentiation. VDR-deficient MC progenitors cultured in vitro show an accelerated development into mature MCs and are more responsive to FcRI-mediated stimulation. It is important that VDR-deficient mice show slightly higher densities of mature MCs in the skin, possibly indicative of a higher rate of differentiation in vivo in the absence of VDR basal activity. These results are not secondary to alterations in calcium blood levels and therefore, support the hypothesis of a physiological involvement of VDR in regulation of MC development. The reported high levels of IgE antibodies circulating in VDR-KO mice [50
] might also play a role in the maintenance of higher MC levels in the skin. In fact, binding of IgE antibodies to FcRI has been shown to regulate cellular function and to promote MC survival, and monomeric IgE is capable of rendering MCs resistant to apoptosis induced by growth factor deprivation, even in the absence of FcRI cross-linking [7
]. The relative contribution of this effect to the increased number of MCs observed in the skin of VDR-deficient mice remains to be determined.
The data we show here add new evidence to several earlier studies showing that 1,25(OH)2D3 can regulate survival and differentiation of various cell lines, including malignant cells [1 , 55 , 56 ] and several immune cell types, comprising dendritic cells (DC) and macrophages [2 , 57 ]. 1,25(OH)2D3 prevents differentiation of monocytes into immature DC and inhibits maturation of DC. Differently from what we show here for MCs, 1,25(OH)2D3 exerts proapoptotic effects mainly on mature DC instead of precursor cells [2 ]. Differently, in bone marrow macrophage precursor cultures with CSF-1, 1,25(OH)2D3 exerts antiproliferative effects and induces macrophage differentiation [57 ].
Here, we have observed that 1,25(OH)2D3 can exert discrete effects on survival and differentiation of MC precursors. In particular, 1,25(OH)2D3 appears to have developmental stage-dependent effects, by inhibiting final maturation of FcRI expressing late MC precursors without affecting their viability and inducing apoptosis of early MC precursors, possibly at the time of the acquisition of FcRI expression. Given the fact that MCs represent a principal effector cell type of Th2 responses, our results add supporting evidence and a novel mechanism by which 1,25(OH)2D3 can inhibit allergic inflammatory responses [48
49
50
, 53
, 54
]. Furthermore, our data support a role for VDR in MC development under physiological conditions and raise the possibility to use 1,25(OH)2D3 analogs in the treatment of MC-related disorders such as mastocytosis. Future investigations will be needed to further clarify the role played by VDR in the regulation of MC maturation and function in physiological as well as pathological settings, such as those associated with allergic responses and parasite infections.
Received May 11, 2006; revised September 1, 2006; accepted September 7, 2006.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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