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(Journal of Leukocyte Biology. 2005;78:605-611.)
© 2005 by Society for Leukocyte Biology

Strain difference of murine bone marrow-derived mast cell functions

Junko Noguchi, Etsushi Kuroda and Uki Yamashita1

Department of Immunology, School of Medicine, University of Occupational and Environmental Health, Japan, Kitakyushu

1Correspondence: Department of Immunology, School of Medicine, University of Occupational and Environmental Health, Japan, 1-1 Iseigaoka, Yahatanishiku, Kitakyushu 807-8555, Japan. E-mail: yama-uki{at}med.uoeh-u.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mast cells play an important role for the induction and the expression of allergic responses. In this report, we studied the strain difference of bone marrow-derived murine mast cell (BMMC) functions in vitro. BMMC were induced by in vitro culture of bone marrow cells from BALB/c and C57BL/6 mice with interleukin (IL)-3 for 4 weeks, stimulated with immunoglobulin E antibody and antigen, and mediators and cytokines released in the culture supernatant were assayed. BMMC from C57BL/6 mice released a higher amount of granule-associated mediators, ß-hexosaminidase, and histamine than that from BALB/c mice. The expression of mRNA of histidine decarboxylase was higher in C57BL/6 mice. Conversely, the productions of newly synthesized mediators, prostaglandin D2 (PGD2), IL-6, and monocyte chemoattractant protein-1, and the mRNA expression of IL-5 were higher in BALB/c BMMC than C57BL/6 BMMC. Although mRNA and protein expression levels of cyclooxygenase-2 were equal in two strains, both expression levels of hematopoietic PGD synthase (hPGDS) were higher in BALB/c BMMC. Mast cells, freshly obtained from mice, also showed the same strain difference concerning the mediator release. These results indicate that the strain difference exists in mast cell functions in mice, and this difference can be considered to induce the susceptibility difference to allergic reactions in mouse strains.

Key Words: rodent • histamine • prostaglandin D2 • cytokine


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mast cells are important effector cells in the expression of allergic responses [1 , 2 ]. After the cross-linking of Fc receptor for immunoglobulin E (IgE; Fc{varepsilon}R) antibody and antigen, they secrete preformed mediators such as histamine, serotonin, and proteoglycans stored in secretory granules and generate lipid mediators such as prostaglandins (PGs), leukotrienes (LTs), and platelet-activating factors, which mediate several clinical symptoms of allergy [3 , 4 ]. Recently, mast cells are reported to work as effector cells for innate immunity and regulator cells for acquired immunity by synthesizing several cytokines and chemokines [5 , 6 ].

Immune responses are regulated with two types of T cell subpopulations, T helper cell type 1 (Th1) and Th2 [7 , 8 ]. Th1 produce mainly interleukin (IL)-2 and interferon-{gamma} and regulate cell-mediated immune responses, and Th2 produce mainly IL-4, IL-5, and IL-10 and regulate humoral immune responses. The balance of Th1/Th2 is changed by an immunization procedure and a type of infection. Th1/Th2 balance is also different in mouse strains. BALB/c mice are the Th2-dominant strain, which easily induce antibody responses after infections and immunizations and are commonly used for the study of allergic responses. C57BL/6 mice are the Th1-dominant strain, which easily induce cell-mediated immune responses [9 , 10 ]. We also reported that macrophage functions were different between BALB/c and C57BL/6 mice [11 ]. Macrophages from BALB/c mice produce a larger amount of PGE2 than that from C57BL/6 mice. These evidences raised the question of whether a strain difference between BALB/c and C57BL/6 mice exists in mast cell functions as effector cells in the allergic responses.

In this study, we show evidence that mast cells from BALB/c mice produce a larger amount of newly synthesized mediators, PGD2, IL-6, and monocyte chemoattractant protein-1 (MCP-1), than from C57BL/6 mice, and mast cells from C57BL/6 mice release a larger amount of granule-associated mediators, ß-hexosaminidase and histamine. These results indicate that the strain difference exists in mast cell functions in mice, and this difference can be considered to induce the susceptibility difference of allergic reactions.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
Female BALB/c and C57BL/6 mice (5–8 weeks old) were purchased from Charles River Japan (Yokohama). These mice were maintained in the Laboratory Animal Research Center at the University of Occupational and Environmental Health, Japan (Kitakyushu) under specific pathogen-free condition. All animal experiments were performed according to the guidelines for the care and use of animals approved by the University of Occupational and Environmental Health, Japan.

Reagents
Recombinant mouse IL-3 was purchased from R&D Systems (Minneapolis, MN). Mouse anti-dansyl (DNS) IgE antibody was purchased from BD Biosciences (San Jose, CA). Anti-cyclooxygenase-2 (COX-2) antibody and anti-hematopoietic PGD synthase (hPGDS) antibody were purchased from Cayman Chemicals (Ann Arbor, MI). DNS-conjugated bovine serum albumin (BSA) was prepared by mixing BSA with 1-dimethylaminonaphthalene-5-sulfonyl chloride (Sigma-Aldrich, St. Louis, MO) at 37°C for 20 h.

Cell preparation and culture
Bone marrow cells were obtained by flushing the femurs of BALB/c and C57BL/6 mice. The lineage-negative cells [CD5–, CD45– receptor (CD45R–), CD11b–, Gr-1–, TER119–, 7/4–] were separated using microbeads (Stem Cell Technologies, Vancouver, Canada). The cells were cultured at 37°C in a humidified atmosphere containing 5% CO2 at a starting density of 5 x 104 cells/ml (1x104 cells/ml for lineage-negative cells) in RPMI-1640 medium (Nissui Pharmaceutical, Tokyo, Japan), supplemented with 5 ng/ml IL-3, 10% fetal bovine serum (FBS; BioWhittaker, Walkersville, MD), 2 mM L-glutamine, 0.1 mM nonessential amino acids, 50 U/ml penicillin, 50 µg/ml streptomycin (all from Life Technologies, Rockville, MD), and 50 mM 2-mercaptoethanol (complete RPMI medium; Nacalai Tesque, Kyoto, Japan) [12 ]. After 4 weeks, the recovered populations were composed of >95% mast cells, as judged by morphology with toluidine blue staining and flow cytometry of the expression of Fc{varepsilon}RI and c-kit.

Peritoneal mast cells were isolated by peritoneal lavage according to the method by Yeatman et al. [13 ]. Briefly, the peritoneal cavity was washed by ice-cold phosphate-buffered saline, and the obtained cells were centrifuged on 72.5% Percoll (Amersham Pharmacia Biotech, Piscataway, NJ) at 300 g for 7 min. The pellets were cultured in complete RPMI medium with 10% FBS at 37°C for 1 h to remove any residual macrophages. Nonadherent cells were used as peritoneal mast cells, which were composed of 85–90% of the resultant cell population as determined by toluidine blue staining.

In vitro stimulation of mast cells
Bone marrow-derived murine mast cell (BMMC; 1x106 cells/ml) were sensitized by incubating for 2 h at 37°C with 0.5 µg/ml anti-DNS IgE antibody in complete RPMI medium with 10% FBS. The cells were washed and then stimulated with 10 µg/ml DNS-BSA for various intervals at 37°C. ß-Hexosaminidase and histamine release assays were performed using 0.1% BSA-Tyrode’s buffer [14 ]. The supernatant and pellet samples were collected for cytokine assay.

Measurement of ß-hexosaminidase
ß-Hexosaminidase assay was conducted according to the method of Razin et al. [14 ]. Supernatant (50 µl) and pellet samples, obtained as described above, were incubated with 50 µl 1 mM p-nitrophenyl-N-acetyl-ß-D-galactosaminide (Sigma-Aldrich), dissolved in 0.1 M citrate buffer, pH 5.0, in a 96-well microtitier plate at 37°C for 1 h. The reaction was stopped with 200 µl/well 0.1 M carbonate buffer, pH 10.5. The plate was read at 405 nm by an enzyme-linked immunosorbent assay (ELISA) reader. The net percentage of ß-hexosaminidase release was calculated as follows: ß-hexosaminidase in supernatant/(ß-hexosaminidase in supernatant+ß-hexosaminidase in pellet) x 100.

Assay of cytokine and chemokine
IL-6 was determined by a standard ELISA as described previously [15 ]. MCP-1 was measured using an immunoassay kit (Biosource International, Camarillo, CA). PGD2 was measured using PGD2-methoxime (MOX) enzyme immunoassay (EIA) kit (Cayman Chemicals). Histamine was measured using histamine EIA kit (SPI-BIO, Bretonneux, France) according to the manufacturer’s protocol.

mRNA isolation and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis
Total RNA was prepared using a commercial RNA isolation kit (AquaPure, Bio-Rad, Hercules, CA). mRNA expression was detected by semiquantitative RT-PCR as described previously [16 ]. The sequence of the sense and antisense primers, product size, and conditions and the number of PCR cycles were as follows: IL-4: 5'-CATCGGCATTTTGAACGAGGTCA-3', 5'-CTTATCGATGAATCCAGGCATCG-3', 240 base pairs (bp), 60 s at 94°C for denaturation, 60 s at 56°C for annealing, and 120 s at 72°C for extension, 30 and 34 cycles; IL-5: 5'-GAAAGAGACCTTGACACAGCTG-3', 5'-GAACTCTTGCAGGTAATCCAGG-3', 277 bp, 60 s at 94°C, 60 s at 50°C, and 120 s at 72°C, 30 and 34 cycles; IL-13: 5'-GACTGCAGTCCTGGCTCTTGC-3', 5'-TGAGTCCACAGCTGAGCC-3', 422 bp, 60 s at 94°C, 60 s at 48°C, and 120 s at 72°C, 30 and 35 cycles; COX-1: 5'-CACTAAGACAGACCCGTCATCTCCA-3', 5'-TGCATGTGGCTGTGGATGTCATCA-3', 499 bp, 60 s at 94°C, 60 s at 55°C, and 120 s at 72°C, 30 and 35 cycles; COX-2: 5'-ACTCACTCAGTTTGTTGAGTCATTC-3', 5'-TTTGATTAGTACTGTAGGGTTAATG-3', 583 bp, 60 s at 94°C, 60 s at 50°C, and 120 s at 72°C, 32 and 36 cycles; hPGDS: 5'-GAATAGAACAAGCTGGACTGG-3', 5'-TGGCAGGGATAGCTTGGACTT-3', 462 bp, 60 s at 94°C, 60 s at 55°C, and 120 s at 72°C, 30 and 35 cycles; histidine decarboxylase (HDC): 5'-CATCAAGCAGCCAGGAGCCAGTCTG-3', 5'-GCACGGTAGCTGGCGAGCACACTG-3', 391 bp, 60 s at 94°C, 60 s at 55°C, and 120 s at 72°C, 25 and 35 cycles; glyceraldehyde 3-phosphate dehydrogenase (G3PDH): 5'-ACCACAGTCCATGCCATCAC-3', 5'-TCCACCACCCTGTTGCTGTA-3', 452 bp, 60 s at 94°C, 60 s at 55°C, and 120 s at 72°C, 20 and 25 cycles. PCR was examined at two different cycles as shown above, six to 10 cycles below the maximum amplification, which is the linear part of the increase of PCR products as determined by a preliminary experiment.

Western blot analysis
Cells were lysed on ice with 100 µl Laemmli sample buffer solution [17 ]. An equal protein concentration (25 µg) from obtained samples was boiled for 5 min, electrophoresed by 5–20% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel, transferred to polyvinylidene difluoride membrane (Bio-Rad), and blotted with anti-COX-2, anti-hPGDS antibody (1/1000), anti-ß-actin (1/10000), and horseradish peroxidase-conjugated secondary antibody (1/2000). The reactive bands were visualized with Lumi-Light Plus Western blotting substrate (Roche Diagnostics, Indianapolis, IN) as a substrate and Fluorochem (Alpha Innotech, San Leandro, CA) as a detector [18 ].

Statistical analysis
Bone marrow and peritoneal cells from two to three mice were pooled for each experiment. All experiments were repeated more than three times, and some representative results are shown in figures. The means and standard deviations (see Figs. 2 3 and 7 ) were from triplicate cultures of one representative experiment. Statistical analyses were performed between BALB/c and C57BL/6 mice using Student’s t-test. A confidence level of <0.05 was considered significant [19 ].



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  		Figure 2. Release of ß-hexosaminidase and histamine from BMMC (1x106 cells/ml), which from BALB/c (open bars) and C57BL/6 (solid bars) mice, was sensitized with 0.5 µg/ml anti-DNS IgE antibody for 2 h at 37°C and then stimulated with 10 µg/ml DNS-BSA for 1 h at 37°C. As a positive control, BMMC were stimulated with 100 ng/ml phorbol myristate acetate (PMA). Supernatant and pellets were collected separately and were assessed for ß-hexosaminidase content by an enzyme activity assay (A, B) and histamine content by EIA (C). *, Significantly higher than other mice.

 


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  		Figure 3. PGD2 and cytokine production by BMMC (1x106 cells/ml), which from BALB/c (open bars) and C57BL/6 (solid bars) mice, was sensitized with 0.5 µg/ml anti-DNS IgE antibody for 2 h and then stimulated with 10 µg/ml DNS-BSA for 1 h. Culture supernatants were collected, and the amount of PGD2 was detected as a PGD2-MOX by an EIA (A). The amount of IL-6 (B) and MCP-1 (C) was detected by standard ELISA. *, Significantly higher than other mice.

 


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  		Figure 7. Peritoneal mast cells freshly obtained from mice show the same tendency as BMMC concerning mediators release. Peritoneal mast cells (1x106 cells/ml) from BALB/c (open bars) and C57BL/6 (solid bars) mice were sensitized with 0.5 µg/ml anti-DNS IgE antibody for 2 h and stimulated with 10 µg/ml DNS-BSA for 1 h. Culture supernatants were collected, and the amount of PGD2 was detected as a PGD2-MOX by an EIA (A). The content of ß-hexosaminidase was assessed by an enzyme activity (B). *, Significantly higher than other mice.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Surface marker of in vitro-induced BMMC
Bone marrow cells from BALB/c and C57BL/6 mice were cultured with IL-3 for 4 weeks, and the morphology and surface marker on the harvested cells were examined by toluidine blue staining and flow cytometry. The harvested cells showed >95% toluidine blue(+). As shown in Figure 1 , Fc{varepsilon}RI(+) and c-kit(+) cells were >95%, suggesting that the obtained cells are composed of almost all mast cells. Although the percentage of Fc{varepsilon}RI(+) and c-kit(+) cells was not different between BALB/c and C57BL/6 BMMC, the profiles for Fc{varepsilon}RI and c-kit expression on BALB/c BMMC show more broad distribution than that on C57BL/6 BMMC.



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  		Figure 1. Surface marker of BMMC. In vitro-induced BMMC from BALB/c and C57BL/6 mice were treated with phycoerythrin (PE)-conjugated anti-Fc{varepsilon}RI or anti-c-kit antibody and were detected by flow cytometry. The results show a fluorescence profile of a representative experiment.

 
Release of ß-hexosaminidase and histamine from BMMC
To investigate the function of BMMC, we sensitized BMMC with anti-DNS-IgE antibody, stimulated with DNS-BSA, and measured the release of preformed mediators, ß-hexosaminidase and histamine, from mast cell secretory granules. As shown in Figure 2A , percent release of ß-hexosaminidase of both strains was similar. However, the total content of ß-hexosaminidase was markedly different in two strains. Accordingly, the amount of release of ß-hexosaminidase by C57BL/6 BMMC was higher than that of BALB/c BMMC (Fig. 2B) . Futhermore, the release of histamine was also higher in C57BL/6 BMMC (Fig. 2C) . These results show that the release of preformed mediators is higher in C57BL/6 BMMC than BALB/c BMMC.

Production of PGD2, cytokines, and chemokines
Next, we investigated the production of newly synthesized mediators, PGD2, cytokines, and chemokines from BMMC, which of both strains, was sensitized with anti-DNS-IgE antibody and then stimulated with DNS-BSA. On the contrary to preformed mediators, BMMC from BALB/c mice produced a larger amount of PGD2 than BMMC from C57BL/6 mice (Fig. 3 ). PGE2 was under the detection limit. IL-6 and MCP-1 [CC chemokine ligand 2 (CCL2)] productions were also higher in BALB/c BMMC than C57BL/6 BMMC. IL-4, IL-5, and IL-13 were under the detection limit.

We assessed mRNA expression of cytokines by RT-PCR. mRNA expressions of IL-4, IL-5, and IL-13 were up-regulated after the stimulation. By a semiquantative RT-PCR analysis, the expression of IL-5 mRNA was higher in BALB/c BMMC than in C57BL/6 BMMC (Fig. 4 ). However, there was no difference in IL-4 and IL-13 mRNA expressions between both strains of mice. Macrophage-derived chemokine (CCL22) and thymus and activation-regulated chemokine (CCL17) mRNA expressions were under the detection limit, even after the stimulation (data not shown). These results suggest that the expression and production of newly synthesized mediators, PGD2, cytokines, IL-5 and IL-6, and chemokines, MCP-1, are higher in BALB/c BMMC than C57BL/6 BMMC.



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  		Figure 4. Expression of cytokine mRNA. BMMC (5x106 cells/ml) from C57BL/6 and BALB/c mice were sensitized with 0.5 µg/ml anti-DNS IgE antibody for 2 h and stimulated with 10 µg/ml DNS-BSA for 4 h, and then total RNA was extracted, and the expression of mRNA was detected by RT-PCR. The numbers of PCR cycles were 30 and 34 for IL-4, 30 and 34 for IL-5, 30 and 35 for IL-13, and 20 and 25 for G3PDH.

 
Expression of COX-1, COX-2, PGD synthase, and HDC
Next, we assessed whether the enhanced productions of PGD2 in BALB/c BMMC and histamine in C57BL/6 BMMC are a result of the difference in mRNA expression of their enzymes. PGH2 is synthesized from arachidonic acid by COX, especially COX-2, and is converted to PGD2 by two PGD synthase isoforms, lipocalin [l] PGDS and hPGDS [20 ]. Mast cell has the latter one. Histamine is synthesized from histidine by HDC [21 ]. As shown in Figure 5 , COX-2, hPGDS, and HDC mRNAs were induced after the antigen stimulation, and COX-1 mRNA was not up-regulated by the stimulation. The expression level of hPGDS mRNA was higher in BALB/c BMMC, and that of HDC was higher in C57BL/6 BMMC. There was no significant difference in COX-2 mRNA expression in two strains of mouse.



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  		Figure 5. Expression of COX-1, COX-2, hPGDS, and HDC mRNA in BMMC (5x106 cells/ml), which from C57BL/6 and BALB/c mice, was sensitized with 0.5 µg/ml anti-DNS IgE antibody for 2 h, stimulated with 10 µg/ml DNS-BSA for 1 h (COX-1 and COX-2) or 4 h (hPGDS, HDC, and G3PDH), and total RNA was extracted, and the expression of mRNA was detected by RT-PCR. The numbers of PCR cycles were 30 and 35 for COX-1, 32 and 36 for COX-2, 30 and 35 for hPGDS, 25 and 35 for HDC, and 20 and 25 for G3PDH.

 
We also examined the protein expression of hPGDS and COX-2 in BMMC. As shown in Figure 6 , COX-2 was induced significantly by the antigen stimulation. However, there is no significant difference of COX-2 expression between BALB/c and C57BL/6 BMMC. On the contrary, hPGDS protein was expressed even before the antigen stimulation and to some extent, decreased after the stimulation. However, the amount of hPGDS was always higher in BALB/c BMMC than in C57BL/6 BMMC. These results indicate that the increased production of PGD2 by BALB/c BMMC is a result of a higher expression of hPGDS enzymes.



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  		Figure 6. Protein expression of COX-2 and hPGDS in BMMC (2.5x106 cells/ml), which from C57BL/6 and BALB/c mice, was sensitized with 0.5 µg/ml anti-DNS IgE antibody for 2 h and stimulated with 10 µg/ml DNS-BSA for 4 h. Then, cells were lysed, electrophoresed, and Western-blotted with anti-COX-2, anti-hPGDS, or anti-ß-actin antibody.

 
Strain difference of peritoneal mast cells
Finally, we assessed whether the strain differences existed in mast cells freshly obtained from mice the same as in vitro-induced BMMC. We prepared mast cells from peritoneal lavage and assessed the content of ß-hexosaminidase and the production of PGD2 after Fc{varepsilon}R-mediated stimulation. As shown in Figure 7 , peritoneal mast cells show the same tendency to BMMC. The content of ß-hexosaminidase is higher in C57BL/6 mast cells, and the generation of PGD2 is higher in BALB/c mast cells. These results suggest that in vitro-induced BMMC reflect the in vivo mast cell functions.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this communication, we studied the strain difference of BMMC functions using BALB/c and C57BL/6 mice, which markedly differ in their Th1/Th2 balance: BALB/c mice are Th2-dominant and C57BL/6 mice are Th1-dominant [9 10 11 ]. As mast cell functions, we examined the release of preformed mediators, newly synthesized mediators, and cytokines after Fc{varepsilon}R cross-linking.

Mast cells were induced in vitro from bone marrow cells by the culture with IL-3 for 4 weeks. The recovered cells were >95% toluidine blue(+), Fc{varepsilon}RI(+), and c-kit(+), suggesting that the induced cells are almost all mast cells. There is no significant difference in the morphology and surface marker of BMMC between BALB/c and C57BL/6 mice. However, BALB/c BMMC showed a more broad distribution profile of Fc{varepsilon}RI and c-kit expression, suggesting that BALB/c BMMC are more heterogeneous concerning Fc{varepsilon}RI and c-kit expression than C57BL/6 BMMC (Fig. 1) .

Functionally, there is a significant difference between BALB/c and C57BL/6 BMMC. BALB/c BMMC produced a larger amount of newly synthesized mediators such as PGD2, IL-6, and MCP-1 than C57BL/6 BMMC (Fig. 3) , and C57BL/6 BMMC released a larger amount of preformed mediators such as ß-hexosaminidase and histamine stored in the granules (Fig. 2) . In accordance with the mediator release, the mRNA and protein expressions of hPGDS and HDC were increased in BALB/c and C57BL/6 mice, respectively (Figs. 5 and 6) . The protein expression of hPGDS was not enhanced by the antigen stimulation but to some extent, decreased, although its mechanism is not clear. However, hPGDS expression was always higher in BALB/c BMMC than C57BL/6 BMMC. The same strain differences of mast cell functions were also obtained in BMMC induced from lineage-negative (CD5–, CD45R–, CD11b–, Gr-1–, TER119–, 7/4–) cells of bone marrow and induced with the combination of IL-3 and stem cell factor (SCF; c-kit ligand; data not shown). Furthermore, mast cells freshly obtained from mice also showed the same tendency in the strain difference as in vitro-induced BMMC (Fig. 7) . These results suggest that the strain difference exists in mast cell functions between BALB/c and C57BL/6 mice.

An interesting finding in this report is that the release of preformed mediators such as ß-hexosaminidase and histamine is not parallel with the production of newly synthesized mediators such as PGD2, IL-6, and MCP-1. Kandere-Grzybowsha et al. [22 ] reported that IL-1 induced IL-6 without degranulation from human mast cells. Gagari et al. [23 ] reported that the SCF induced IL-6 without histamine release. These reports and our findings suggest that the degranulation and the release of newly synthesized mediators are different phenomena and are controlled by different mechanisms.

In our previous paper, we reported that the strain difference existed in PGE2 production by macrophages between BALB/c and C57BL/6 mice, and this difference was induced by the difference of mPGES level, enzyme converting PGH2 to PGE2 [11 ]. Mast cells produced PGD2, but not PGE2, and the level of PGD2 in mast cells also showed the same strain difference of mice as the level of PGE2 in macrophages. Other prostanoid product is LTC4. Okayama et al. [6 ] reported that LTC4 production is parallel with that of PGD2, and mast cells produced a larger amount of PGD2 than LTC4. Therefore, we studied only PGD2 production in this report.

Many clinical symptoms of type I allergy, such as rhinitis and conjunctivitis induced by vasodilatation and increased venular permeability, asthmatic attack induced by smooth muscle cell contraction, and mucus reaction, are induced by histamine and histamine receptors [3 , 4 ]. However, some of them are not relieved by antihistamine drugs [24 ]. Recently, the importance of PGD2 is indicated as a mediator of allergic responses. Matsuoka et al. [25 ] reported that PGD receptor-deficient mice showed the reduced level of Th2 cytokines and lymphocyte accumulation in the lung and failed to develop airway hypersensitivity. Honda et al. [26 ] reported that PGD2 reinforced Th2 inflammatory responses of airways. Gosset et al. [27 ] and Hammad et al. [28 ] reported that PGD2 affected the maturation of monocyte-derived dendritic cells. Hirai et al. [29 ] reported that PGD2 induced chemotaxis of Th2 cells. Furthermore, cytokines and chemokines such as IL-6 and MCP-1 play an important role for the induction of inflammation and cell migraion [22 , 23 , 30 ].

In conclusion, we found that mast cells from BALB/c mice produced a larger amount of newly synthesized mediators such as PGD2, IL-6, and MCP-1 than mast cells from C57BL/6 mice, and mast cells from C57BL/6 mice released a larger amount of preformed mediators such as ß-hexosaminidase and histamine than mast cells from BALB/c mice. This is the first report to show the existence of strain difference of murine mast cell functions. Mast cells work for not only effector cells for allergic responses but also regulator cells for the induction of an acquired immunity [5 , 6 ]. BALB/c and C57BL/6 mice are representative Th2- and Th1-dominant strains, respectively, and these Th1- and Th2-dominant responses in each mouse strain are explained by the polarization of T cells and macrophages [9 10 11 ]. Our results suggest that mast cells also participate in the determination of Th1 and Th2 responses.


   ACKNOWLEDGEMENTS
 
This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (15590444).

Received November 21, 2004; revised May 9, 2005; accepted May 10, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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