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The Journal of Immunology, 2007, 178: 4177-4183.
Copyright © 2007 by The American Association of Immunologists, Inc.

Fc{epsilon}RI Aggregation Promotes Survival of Connective Tissue-Like Mast Cells but Not Mucosal-Like Mast Cells1

Maria Ekoff*, Andreas Strasser{dagger} and Gunnar Nilsson2,*

* Department of Medicine, Clinical Immunology and Allergy Unit, Karolinska Institutet, Stockholm, Sweden; and {dagger} Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Mast cells play a critical role in IgE-dependent immediate hypersensitivity reactions. This is facilitated by their capacity to release inflammatory mediators and to undergo activation-induced survival upon cross-linking of the high-affinity IgE-receptor (Fc{epsilon}RI). Due to their heterogeneity, mast cells can be divided into two major groups: the connective tissue mast cells and the mucosal mast cells. We have previously shown that IL-3-dependent bone marrow-derived mast cells can undergo activation-induced survival that is dependent on the prosurvival gene A1. In this study, we have used two different protocols to develop murine connective tissue-like mast cells (CTLMC) and mucosal-like mast cells (MLMC) to investigate their capacity to survive an allergic reaction in vitro. In this study, we demonstrate that Fc{epsilon}RI stimulation promotes survival of CTLMC but not MLMC. Similarly, a prominent induction of A1 is observed only in CTLMC but not MLMC. MLMC have a higher basal level of the proapoptotic protein Bim compared with CTLMC. These findings demonstrate a difference among mast cell populations in their ability to undergo activation-induced survival after Fc{epsilon}RI stimulation, which might explain the slower turnover of CTMC in IgE-dependent reactions.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Mast cells are of hemopoietic origin, derived from CD34+ progenitor cells in the bone marrow (1). As undifferentiated mononuclear cells, the mast cells are recruited from peripheral blood into the tissue where they mature, under the influence of stem cell factor (SCF)3 and locally produced cytokines, into multifunctional effector and regulatory cells of the immune system (2, 3). Mast cells do not necessarily represent a homogenous population, since differentiation and maturation of mast cells are influenced by the microenvironment, which results in morphological, biochemical, and functional differences (4). Human mast cells are classified into two types on the basis of the expression of proteases in their granules: mast cells containing tryptase (MCT) and mast cells containing tryptase and chymase (MCTC) (5). Rodent mast cells can be divided into two types, based on their tissue distribution; connective tissue mast cells (CTMC) and mucosal mast cells (MMC) (6). In terms of tissue localization and T cell responsiveness, human MCT correspond to rodent MMC, being located predominantly in mucosal tissues, such as the intestine and respiratory tract, whereas human MCTC correspond to rodent CTMC and are mainly found in connective tissues, such as the skin and peritoneal cavity (7). Furthermore, based on the expression of mast cell-specific proteases, additional subpopulations have been identified in vivo (8).

One characteristic feature of mast cells is their longevity; situated in the tissue they can survive for several months. Although MMC are still capable of proliferating, their number is kept relatively constant under normal conditions. However, the number of MMC increases remarkably during inflammation due to the action of T cell-derived cytokines (9, 10, 11). CTMC appear not to depend on T cell-derived cytokines but primarily require SCF for their persistence (2, 12). Inhibition of SCF production in tissues leads to increased mast cell apoptosis and the in vitro development of human mast cells requires SCF (3, 13).

Mast cells are known for their ability to mediate IgE-dependent responses. Cross-linking of the high-affinity receptor Fc{epsilon}RI on mast cells leads to aggregation and subsequent activation. This typically results in degranulation, changes in gene expression, and the release of inflammatory mediators (3, 14). The cross-linking of Fc{epsilon}RI has also been shown to affect mast cell survival (15, 16, 17). Central regulators of mast cell survival and apoptosis are the members of the Bcl-2 protein family. The Bcl-2 family consists of pro- and antiapoptotic members characterized by Bcl-2 homology (BH) domains. The proapoptotic members can be further divided into two subgroups: BH3 domain-only proteins (BH3-only) and Bax/Bak-like proteins (18, 19). In response to developmental signals or experimentally applied stress stimuli, BH3-only proteins are activated by transcriptional and/or posttranslational mechanisms (20). They then trigger apoptosis by binding, in a selective fashion, to prosurvival Bcl-2 family proteins, which leads to the activation of Bax and Bak causing activation of the caspase cascade and, ultimately, cell destruction.

The proapoptotic Bcl-2-interacting mediator of cell death, Bim (also called Bod), belongs to the BH3-only subgroup of the Bcl-2 family. Studies with gene-targeted mice have shown that Bim is essential for normal regulation of apoptosis in numerous cell types, including B and T lymphocytes, monocytes, granulocytes, and neurons (21, 22, 23, 24). We have previously shown that SCF regulates mast cell survival through phosphorylation of Bim, which primes this protein for ubiquitination and proteasomal degradation (25). Mast cells lacking Bim are less sensitive to apoptosis upon growth factor (e.g., SCF) deprivation than wild-type (wt) mast cells (26). Collectively, these findings indicate an important role for Bim in regulating mast cell apoptosis. In contrast, Fc{epsilon}RI stimulation-induced mast cell survival is regulated by the anti-apoptotic Bcl-2 family member A1 (17). We have demonstrated that mRNA levels for A1 are increased following Fc{epsilon}RI aggregation and that mast cells lacking A1 do not gain a survival advantage from Fc{epsilon}RI cross-linking (17). Similarly, the human homolog of A1, Bfl-1, is up-regulated in human mast cells upon Fc{epsilon}RI aggregation (27). These observations show that A1 is critical for Fc{epsilon}RI-mediated activation-induced mast cell survival.

Previous studies on in vitro-derived murine mast cells have mainly been performed on mast cells derived from bone marrow cells cultured in the presence of IL-3, i.e., bone marrow-derived mast cells (BMMC). These cells have been thought to share many features with MMC (28). However, it has subsequently been shown that these BMMC are not morphologically similar to MMC and they express only few or none of the chymases typically found in MMC (29). Attempts to generate MMC in vitro have been made and using a combination of SCF, IL-3, IL-9, and TGF-beta1, Miller et al. (30, 31) differentiated murine bone marrow cells into mast cells expressing the MMC-specific chymase mouse MC protease (mMCP) 1 within 1 wk. In this system, TGF-beta1 appears to be the key factor promoting the development of MMC-like BMMC. In contrast, nerve growth factor and SCF can induce the differentiation of BMMC into a population resembling CTMC (32, 33). Moreover, IL-4 acts in synergy with IL-3 and SCF inducing proliferation of BMMC in culture and it also helps in maintaining the CTMC characteristics of mouse peritoneal mast cells in vitro (34, 35, 36, 37).

We have taken advantage of the current knowledge to produce murine MLMC and CTLMC to further characterize these in vitro-developed mast cell populations. These cells were used to analyze activation-induced cell survival in different mast cell phenotypes and we show that CTLMC but not MLMC exhibit Fc{epsilon}R stimulation-induced up-regulation of the prosurvival gene A1 and enhanced cell survival.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

The mice used were Bim-deficient (266/266del) mice backcrossed for >12 generations onto a C57BL/6J genetic background and wt C57BL/6J mice (from the Walter and Eliza Hall Institute mouse breeding facility, Kew, Australia, and the Karolinska Institute mouse breeding facility at the Department of Microbiology, Tumor and Cell Biology, Stockholm, Sweden). All experiments with animals were performed according to the guidelines of the Royal Melbourne Hospital Research Foundation Animal Ethics Committee and the Animal Ethics Committee in Stockholm.

Cell cultures

BMMC were differentiated into either a connective tissue-like phenotype (CTLMC) or a mucosal-like phenotype (MLMC). CTLMC characteristics were obtained by culturing mouse bone marrow cells in RPMI 1640 medium containing 10% filtered FCS, 4 mM L-glutamine, 1 mM sodium pyruvate, 100 IU/ml penicillin G, 100 µg/ml streptomycin, 0.1 mM MEM nonessential amino acids, and 50 µM 2-ME (Sigma-Aldrich), supplemented with either 50 or 25 ng/ml recombinant murine SCF (recombinant murine SCF, obtained from either Amgen or produced in Pichia pastoris and purified from culture supernatants by reverse-phase chromatography (provided by Dr. H. Martin, Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia)) and 1 ng/ml murine rIL-4 (PeproTech). Alternatively, MLMC were produced in DMEM containing IL-3 (5% supernatant of X63/0 myeloma cells transfected with an IL-3 expression vector), 10% filtered FCS, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 IU/ml penicillin G, 100 µg/ml streptomycin, and supplemented also with either 50 or 25 ng/ml recombinant murine SCF, 5 ng/ml recombinant murine IL-9, and 1 ng/ml recombinant human TGF-beta1 (PeproTech). All cells were cultured for a minimum of 2 wk before they were used. The maturity and purity of the cells were examined by toluidine blue staining and flow cytometric analysis for expression of Kit and Fc{epsilon}RI, using FITC-anti-mouse CD117 (Kit) mAb 2B8 or FITC-conjugated rat IgG2b isotype control (both from BD Pharmingen), FITC-conjugated anti-mouse Fc{epsilon}RI-{alpha} mAb MAR-1, or FITC-conjugated Armenian hamster IgG isotype control (both from eBioscience). To monitor apoptosis, cells were stained with propidium iodide (2 µg/ml) plus FITC-conjugated annexin V (0.3 µg/ml) and analyzed in a FACScan (BD Biosciences). Cell proliferation was measured using a [3H]thymidine ([3H]TdR) incorporation assay (38) exposing cells to [3H]TdR (37 kBq/well) for 7 h of culture.

Mast cell activation

For activation through cross-linking of the IgE receptor, mast cells were initially sensitized for 90 min with a monoclonal mouse anti-trinitrophenyl (TNP) IgE Ab (IgEl-b4; American Type Culture Collection) used as a 15% hybridoma supernatant. Cells to be used in the N-acetyl-beta-D-hexosaminidase assay were then subjected to two washings with PBS and resuspended in RPMI 1640 medium supplemented with 0.2% BSA (Sigma-Aldrich) before the cells were activated by the addition of 100 ng/ml TNP-BSA (Biosearch Technologies) with a coupling ratio of 9:1. In experiments where activation-induced survival after Fc{epsilon}RI cross-linking was measured, the mast cells were deprived of cytokines and kept in medium supplemented with 10% FCS during sensitization and activation as previously described. For activation using compound 48/80, cells were kept in RPMI 1640 medium supplemented with 0.2% BSA (Sigma-Aldrich) and activated using 5 µg/ml compound 48/80 (Sigma-Aldrich) before being used in the N-acetyl-beta-D-hexosaminidase assay. Cells were cultured at a concentration of 106 cells/ml.

N-acetyl-beta-D-hexosaminidase release assay

For detection of the granular enzyme beta-hexosaminidase, an enzymatic colorimetric assay was used as described previously (17). Briefly, 60 µl of supernatant was transferred to a 96-well plate and mixed with an equal volume of substrate solution (7.5 mM p-nitrophenyl-N-acetyl-beta-D-glucosaminide dissolved in 80 mM citric acid, pH 4.5). The mixture was incubated on a rocker platform for 2 h at 37°C. After incubation, 120 µl of glycine (0.2 M, pH 10.7) was added to each well, and the absorbance at 405 and 490 nm was measured using an Emax Precision Microplate Reader (Molecular Devices).

Enzyme immunoassay for histamine

Cells were resuspended in distilled water and lysed by two rapid freeze/thaw cycles before being used. Histamine in the pellet was quantitated by using the immunoassay histamine EIA IM2015 (Beckman Coulter) according to the manufacturer’s protocol and an Emax Precision Microplate Reader (Molecular Devices).

RT-PCR

Total RNA was extracted from cell pellets in 1 ml of TriPure isolation reagent (Roche Diagnostics) according to the manufacturer’s protocol. RNA was reverse-transcribed into single-stranded cDNA using a First Strand cDNA Synthesis Kit according to the manufacturer’s protocol (Roche Diagnostics). The cDNA was amplified with the PCR Core Kit (Roche Diagnostics) using two pairs of oligonucleotide primers to identify transcription of chymase genes commonly expressed in mouse mast cells: mMCP-1 (460 bp) 5' primer, 5'-GGAAAACTGGAGAGAAAGAACCTAC and 3' primer, 5'-GACAGCTGGGGACAGAATGGGG (30) and mMCP-5 (562 bp) 5' primer, 5'-AGGAGCCCATAACAAAACAT and 3' primer, 5'-TATTCCAGTTCCAGATTTCC. GAPDH was used as a control of cDNA integrity. In brief, the cDNA was amplified for 30 cycles in a thermal cycler (Gene Amp PCR system 2400; PerkinElmer), one cycle being composed of denaturation at 94°C for 30 s, annealing at 63°C (mMCP-1) or 54°C (mMCP-5) for 45 s, and elongation at 72°C for 1 min. PCR products were separated on 2% agarose gels containing 0.5 µg/ml ethidium bromide and visualized and recorded under UV light.

RNase protection assay (RPA)

RPA was performed according to the RiboQuant System protocol using a custom-made multiprobe set (BD Pharmingen). The gel was dried and exposed on Kodak film (Eastman Kodak) with intensifying screens at –70°C. Expression of RNA was detected with a phosphor imager device and levels of expression were quantified using MacBas version 2.2 software (Fuji Photo Film).

Statistical analysis

We used either the Student t test or an ANOVA followed by multiple comparison with the Wilcoxon-matched pairs test or the Kendall coefficient of concordance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Characterization of in vitro-produced MLMC and CTLMC

We first compared the development and phenotype of mast cells cultured in either SCF/IL-4 or SCF/IL-3/IL-9/TGF-beta1, conditions that promote development into either CTLMC or MLMC, respectively. Cultures were analyzed each week with respect to cell number and percentage of mast cells. CTLMC (SCF/IL-4) cultures showed no overall increase in cell number (Fig. 1A), and by week 4 the total cell number even declined to some extent, although this was not associated with a decline in the percentage or total number of mast cells (Fig. 1B). In contrast, MLMC cultures (SCF/IL-3/IL-9/TGF-beta1) showed a decrease in total cell number at week 2 and this was associated with an increase in the percentage of mast cells (Fig. 1, C and D). Cell counts for the two types of culture conditions were similar at week 1 but at week 2 the cell numbers were significantly higher in CTLMC cultures compared with MLMC cultures. However, at weeks 3 and 4, this was reversed with a dramatic increase of total cellularity with mast cells constituting 94–96% of the MLMC cultures. Overall, the cultures grown in SCF/IL-3/IL-9/TGF-beta1-supplemented medium, giving rise to MLMC, showed a more rapid increase in mast cell numbers and also displayed more vigorous proliferation compared with CTLMC cultures (Fig. 1E).


Figure 1
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  		FIGURE 1. Growth and maturation of mast cells cultured under different conditions. CTLMC (SCF/IL-4) cultures showed no overall increase in number of cells as determined by trypan blue staining and cell counting (A). By 4 wk the cell number declined to some extent but this was not associated with a decline in the percentage or total numbers of mast cells (B). MLMC (SCF/IL-3/IL-9/TGF-beta1) cultures showed a decrease in total cell number after 2 wk and this was associated with an increase of the percentage of mast cells as determined by toluidine blue staining (C and D, respectively). Proliferative response for pure cultures of CTLMC (SCF/IL-4) and MLMC (SCF/IL-3/IL-9/TGF-beta1) as determined by [3H]thymidine incorporation 7 h; (E). n = 3 and results are presented as mean ± SD. **, p < 0.01.

 
Changes in morphology could be observed by staining the differentiating mast cells with toluidine blue each week (Fig. 2A). After 1 wk of culture in SCF/IL-4 (CTLMC), only a small portion, ~20%, of the cells stained positive with toluidine blue and appeared to be mast cells (Fig. 2A, left upper panel). In contrast, at this time, nearly 40% of the cells cultured in SCF/IL-3/IL-9/TGF-beta1 (MLMC) already displayed the phenotypical characteristics of mast cells and were stained with toluidine blue (Fig. 2A, left lower panel). Sequential changes in morphology could be observed for CTLMC cultures when these cells gradually became more toluidine blue positive. By week 4 the cytoplasm of the cells mostly contained granules that were stained with toluidine blue as strongly as cells from MLMC cultures (Fig. 2A, right upper and lower panels, respectively). Comparing the mast cells grown in either SCF/IL-4 or SCF/IL-3/IL-9/TGF-beta1-supplemented medium, it was evident that they display differences in morphology and progression of differentiation. MLMC cultures grown in SCF, IL-3, TGF-beta1, and IL-9 exhibited more rapid progression of differentiation, fewer toluidine-positive cytosolic granules, and the mast cells also displayed a somewhat less structured surface.


Figure 2
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  		FIGURE 2. Differences in morphology and progression of differentiation of mast cells at week 1 (A, left panels) and week 4 (A, right panels), respectively, for CTLMC cultures (SCF/IL-4) (A, upper panels) or MLMC cultures (SCF/IL-3/IL-9/TGF-beta1) (A, lower panels). CTLMC and MLMC both express the receptors c-Kit and Fc{epsilon}RI on their surface, as examined at week 4 for CTLMC cultures (SCF/IL-4) (B, upper panels) or MLMC cultures (SCF/IL-3/IL-9/TGF-beta1) at week 2 (B, lower panels). Filled histograms = staining with an isotype-matched control Ab. Thick line = staining with Abs to c-Kit or Fc{epsilon}RI, respectively. Analysis of the transcription of the genes encoding the mast cell chymases mMCP-1 and mMCP-5. No mMCP-1 was detected in CTLMC cultures but mMCP-5 could readily be detected in these cultures (C, lanes 1 and 2, respectively). Levels of both mMCP-1 and mMCP-5 were readily detected in MLMC (C, lanes 3 and 4, respectively). Probing for GAPDH mRNA was used as an internal control (all lanes). The results shown are representative of at least three independent experiments for each cell type. Differences in activation of CTLMC and MLMC upon compound 48/80 treatment as shown by beta-hexosaminidase release (D). n = 3 and results are presented as mean ± SD. **, p < 0.01.

 
To further characterize the in vitro-developed MLMC and CTLMC, we examined their expression of Kit and the high-affinity IgE receptor Fc{epsilon}RI. This was done at week 2 for SCF/IL-3/IL-9/TGF-beta1 cultures (MLMC) and at week 4 for SCF/IL-4 cultures (CTLMC), the time points when these cultures consisted mostly (>90%) of mast cells. Immunofluorescent staining with specific Abs and flow cytometric analysis demonstrated that the two mast cell populations developed in the two different systems both expressed the receptors Kit and Fc{epsilon}RI (Fig. 2B).

Mast cell subpopulations can be distinguished by their pattern of chymase expression. For example, mMCP-1 is a chymase expressed exclusively in mucosal mast cells, whereas mMCP-5 is found in both CTMC as well as in MLMC grown in SCF, IL-3, TGF-beta1, and IL-9 (30). We therefore analyzed the transcription of the genes encoding the mast cell chymases mMCP-1 and mMCP-5 by week 2 in MLMC (SCF/IL-3/IL-9/TGF-beta1) and by week 4 in CTLMC (SCF/IL-4) cultures. Although readily detectable levels of both mMCP-1 and mMCP-5 could be seen in MLMC, only mMCP-5 was found in CTLMC (Fig. 2C). There was no visible difference in levels of transcription of mMCP-5 comparing the two sets of culture conditions.

The ability of MMC and CTMC to be activated by the well-known mast cell secretagogue compound 48/80 is known to differ, with degranulation occurring in CTMC, but not in MMC (39, 40, 41). When testing our in vitro-developed MLMC and CTLMC, we found that CTLMC but not MLMC could be activated by 5 µg/ml compound 48/80 with degranulation as a result of activation (Fig. 2D). We also determined the total amount of histamine content of MLMC and CTLMC and found the histamine content to be 0.09 ± 0.04 and 4.09 ± 2.34 pg/cell, respectively (mean ± SD and n = 3). This finding is consistent with previous reports showing that CTMC contain more histamine than MMC (42).

Bcl-2 family mRNA expression in MLMC and CTLMC

Members of the Bcl-2 family of proteins are critical regulators of apoptosis and consist of both members promoting apoptosis, as well as proteins that safeguard cell survival (43). The pro- and antiapoptotic members of the Bcl-2 family can heterodimerize and titrate each other’s functions, which suggests that the relative concentrations of these proteins might determine cell fate (44). We therefore investigated whether there are any differences in the expression pattern of Bcl-2 family members comparing MLMC and CTLMC cultures. RPA analysis and quantification by densitometric analysis detected no significant difference in mRNA expression for Bcl-2 family members between MLMC and CTLMC, apart from (~1.5- to 2-fold) increased levels of bim mRNA seen in MLMC (Fig. 3).


Figure 3
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  		FIGURE 3. Resting MLMC contain more bimEL mRNA than resting CTLMC. RPA was performed to analyze and quantitate the expression levels of a set of Bcl-2 family members in CTLMC (SCF/IL-4) and MLMC (SCF/IL-3/IL-9/TGF-beta1) (A). The arrow indicates bimEL. Quantification of transcript levels are shown relative to their levels of GAPDH mRNA and showed differences in bimEL mRNA levels, with MLMC containing more bimEL than CTLMC (B). n = 4 and results are presented as mean ± SD. *, p < 0.05.

 
Fc{epsilon}RI cross-linking promotes only survival of CTLMC but not MLMC

Fc{epsilon}RI cross-linking has been shown to promote survival of mast cells cultured in the absence of their requisite growth factors (16, 17, 45). However, we have previously noted that the culture conditions affect Fc{epsilon}RI activation-induced mast cell survival (26). Therefore, we compared the responses of MLMC (SCF/IL-3 IL-9/TGF-beta1) and CTLMC (SCF/IL-4) to Fc{epsilon}RI cross-linking after growth factor withdrawal. When MLMC were deprived of growth factors, no survival effect was achieved by Fc{epsilon}RI cross-linking. In contrast, Fc{epsilon}RI cross-linking clearly prolonged the survival of growth factor-deprived CTLMC (Fig. 4A). We have previously observed that both proapoptotic (Bim) as well as antiapoptotic (Bcl-xL, A1) members of the Bcl-2 family are up-regulated upon Fc{epsilon}RI cross-linking (17, 26), and we have therefore postulated that mast cell fate is determined by the balance of these proteins (46). This observation and the elevated levels of bim present in MLMC compared with CTLMC at resting state prompted us to examine the in vitro survival of MLMC and CTLMC lacking Bim. We found that when MLMC lacking Bim were deprived of growth factors, these cells survived better compared with wt cells. However, Fc{epsilon}RI cross-linking conferred no additional survival effect (Fig. 4B). In CTLMC, loss of Bim also increased survival after growth factor withdrawal and although the effect of Fc{epsilon}RI cross-linking was somewhat diminished, Fc{epsilon}RI cross-linking was still able to significantly enhance survival (Fig. 4B).


Figure 4
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  		FIGURE 4. Growth factor deprived wt CTLMC but not MLMC survive better after Fc{epsilon}RI cross-linking (A) MLMC from bim–/– mice survived growth factor deprivation better than wt MLMC but they did not show increased survival upon Fc{epsilon}RI cross-linking compared with nonactivated cells (B). Cells were sensitized with 15% IgE before challenge with 100 ng/ml TNP and kept in medium lacking growth factors for 24 h before being analyzed by flow cytometry, using propidium iodide plus FITC-conjugated annexin V. n = 4–6 and results are presented as mean ± SD. *, p < 0.05.

 
Fc{epsilon}RI cross-linking promotes A1 mRNA up-regulation only in CTLMC but not in MLMC

A1 is an anti-apoptotic Bcl-2 family member known to be induced by inflammatory cytokines and Ag receptor ligation, suggesting a possible role of A1 in inflammatory processes (47). Previous work has shown that Fc{epsilon}RI aggregation strongly induced A1 mRNA levels in mast cells (17). Moreover, mast cells lacking A1 did not survive after Fc{epsilon}RI stimulation, demonstrating that A1 is crucial for the ability of mast cells to survive an allergic activation (17). We therefore performed RPA to examine the levels of A1 mRNA in MLMC and CTLMC, both before and after Fc{epsilon}RI cross-linking. Only a slight up-regulation of A1 mRNA was seen in MLMC (SCF/IL-3 IL-9/TGF-beta1) after Fc{epsilon}RI cross-linking, although A1 is present in both resting and activated cells. In contrast, in CTLMC Fc{epsilon}RI cross-linking potently (~6-fold) up-regulated A1 mRNA levels, and, as previously described for BMMC (26), the levels of bimEL also increased slightly (Fig. 5).


Figure 5
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  		FIGURE 5. A1 mRNA expression is induced by Fc{epsilon}RI cross-linking in CTLMC but not in MLMC. RPA was performed on RNA isolated from mast cells sensitized with 15% IgE before challenge with 100 ng/ml TNP for 6 h. The arrow indicates A1 mRNA. Quantification of the expression levels of a set of Bcl-2 family members is shown relative to their levels of GAPDH mRNA. n = 3 and results are presented as mean ± SD. *, p < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
There is considerable heterogeneity among tissue mast cells when comparing their phenotype, function, and survival (7, 48). Using different combinations of cytokines, it is possible to differentiate murine bone marrow stem cells into either MLMC (SCF, IL-3, IL-9, and TGF-beta1) (30) or CTLMC (SCF and IL-4) (37). In this study, we used these culture conditions to further characterize and compare these different mast cell populations to establish their usefulness in mast cell research. We were particularly interested in comparing their responses to Fc{epsilon}RI cross-linking in terms of activation-induced survival. In humans, MMC (MCT) are increased during allergic rhinitis and increased numbers of these cells have also been associated with asthma (49, 50, 51, 52). There is a local accumulation of mucosal mast cells upon Ag exposure in seasonal allergic rhinitis and these cells were also shown to bear IgE on their surface. In contrast, connective tissue mast cells (MCTC) are not increased during allergic rhinitis (49).

We found that in vitro-derived MLMC show a dramatic increase in cell numbers and also displayed more vigorous proliferation, compared with CTLMC. During nematode infections in the gut, mast cells differentiate, become activated and hyperplasia occurs before their numbers decline as the infection resolves (2, 53, 54). MLMC express the beta-chymase mMCP-1 known to be selectively expressed in MMC (55, 56) and the kinetics of the MLMC maturation closely resembles mast cell hyperplasia seen upon nematode infections. In addition, other characteristic differences seen for MMC and CTMC, such as reactivity to compound 48/80 and histamine content, are also seen in our MLMC and CTLMC demonstrating the relationship of these cells with MMC and CTMC. These observations suggest that MLMC and CTLMC represent a useful in vitro model for committed mast cell lineages.

Overall, MMC have a very different life span and growth rate compared with CTMC, which normally are nonproliferative and have a slow turnover rate (57). Our in vitro-derived MLMC and CTLMC were found to display the characteristic differences in proliferation and turnover rate of MMC and CTMC in vivo. To investigate whether the culture conditions leading to differences in proliferation and turnover rate of mast cells affect levels of pro- or anti-apoptotic Bcl-2 family members, we performed RPA. We found that MLMC contained more bimEL compared with CTLMC. Interestingly, this difference in the levels of a proapoptotic Bcl-2 protein coincides with the MLMC having a very high proliferation rate and one could speculate that the elevated level of Bim could be due to the cells being subjected to stress, since the nonproliferating CTLMC display a lower level of bimEL. We have previously also observed that culture conditions can affect Fc{epsilon}RI activation-induced mast cell survival (26) and we therefore compared the survival of MLMC and CTLMC after exposure to Fc{epsilon}RI cross-linking and growth factor withdrawal. Strikingly, MLMC deprived of growth factors did not respond to Fc{epsilon}RI cross-linking with increased survival, whereas CTLMC did. MLMC do express the high-affinity IgE receptor Fc{epsilon}RI and are clearly responding to Fc{epsilon}RI cross-linking as evidenced by degranulation (data not shown).

Our previous work indicated that the anti-apoptotic Bcl-2 family member A1/Bfl-1 is critical for Fc{epsilon}RI cross-linking-induced mast cell survival (27). Interestingly, A1 is present both in resting and stimulated MLMC, but only a slight up-regulation was observed in A1 mRNA after Fc{epsilon}RI cross-linking. In contrast, Fc{epsilon}RI cross-linking of CTLMC caused a profound up-regulation of A1. Furthermore, MLMC had a higher basal level of proapoptotic bim compared with CTLMC.

The BH3-only protein Bim has a critical role since it can bind with high affinity to all anti-apoptotic Bcl-2 family members, including A1, which appears to have an important antiapoptotic function in mast cells (17). It has recently been reported that the interaction between A1 and Bim stabilizes the A1 protein, thereby strongly reducing A1 turnover and amplifying its antiapoptotic effects (58). However, Bim is not the only BH3-only protein that can bind to A1, e.g., Puma can also do this (59). Because anti-apoptotic Bcl-2 family members, such as A1, interact with BH3-only proteins to regulate cell fate, one could speculate that A1 up-regulation counteracts the higher expression levels of bimEL in CTLMC after Fc{epsilon}R cross-linking. The differences in the ability to increase A1 expression might explain, at least in part, the differences in survival and turnover of CTMC and MMC in vivo. Collectively, these data suggest that the coinduction of A1 and Bim might balance each other and determine whether the mast cell will survive or undergo apoptosis. Knowledge of the binding of A1 to pro-apoptotic Bcl-2 family proteins in mast cells is limited and its subsequent impact on regulation of mast cell survival and apoptosis is currently under investigation.


   Acknowledgments
 
We thank Dr. H. Martin and P. Morgan for gifts of SCF; Dr. H. Karasuyama for IL-3-producing cells; Drs. L. O’Reilly and D. Huang for Abs; and Dr. P. Bouillet and Profs. J. Adams, and S. Cory for providing the bim–/– mice.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
G. Nilsson has a patent based on A1 in mast cells.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the Swedish Research Council-Medicine; the Swedish Cancer Foundation, Cancer and Allergy Foundation; Ellen, Walter and Lennart Hesselmans Foundation for Scientific Research; Consul Th C Berghs Foundation; Ollie and Elof Ericsson’s Foundation; and King Gustav V’s 80-year foundation (all from Sweden); the National Health and Medical Research Council (Canberra, Australia, Grant 257502); the JDRF/National Health and Medical Research Council (Australia); the National Cancer Institute (National Institutes of Health Grants CA80188 and CA43540); and the Leukemia and Lymphoma Society of America (Specialized Center of Research Grant 7015). Back

2 Address correspondence and reprint requests to Dr. Gunnar Nilsson, Department of Medicine, Karolinska Institutet, Clinical Immunology and Allergy Unit, KS L2:04, Stockholm, Sweden. E-mail address: gunnar.p.nilsson{at}ki.se Back

3 Abbreviations used in this paper: SCF, stem cell factor; MCT, mast cells containing tryptase; MCTC, MC containing tryptase and chymase; CTMC, connective tissue MC; MMC, mucosal MC; BMMC, bone marrow-derived MC; BH, Bcl-2 homology; wt, wild type; Bim, Bcl-2 interacting mediator of cell death; CTLMC, connective tissue-like MC; MLMC, mucosal-like MC; mMCP, mouse MC protease; RPA, RNase protection assay. Back

Received for publication June 2, 2006. Accepted for publication January 22, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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