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Sixteen heat-killed bifidobacteria isolated from human intestine and a probiotic strain Lactobacillus GG were tested for their ability to influence IgE-mediated degranulation of rat basophilic leukemia (RBL-2H3) cells in vitro. The bifidobacteria suppressed IgE-mediated degranulation of RBL-2H3 cells by 1.6–56.4% in a strain-dependent manner. Bifidobacteria from healthy infants expressed high inhibitory effects on IgE-mediated degranulation (41–55%), while those from allergic infants varied greatly in their effects against degranulation. Bifidobacteria taxonomically identified as Bifidobacterium bifidum exhibited much stronger inhibitory effects against IgE-mediated degranulation than those taxonomically identified as B. adolescentis (P < 0.05).These results indicate that the intestinal bifidobacteria might be one of factors influencing IgE-mediated allergic responses.
Bifidobacteria are one of the main groups of human intestinal microbiota which are predominant in infants (1). Low numbers of intestinal bifidobacteria have been reported to be an important aspect of aberrant intestinal microbiota in allergic infants (2–4), and these low numbers of Bifidobacterium have been detected even before the onset of allergy (2). In addition to these quantitative differences in Bifidobacterium microbiota, qualitative differences were also observed in our previous studies (5, 6). B. adolescentis and B. longum were the predominant bifidobacteria isolated from allergic infants, whereas the predominant bacteria isolated from age-matched healthy infants were B. infantis, B. bifidum, and B. breve. Recently, more scientific evidence has been reported which supports the findings that, in comparison to infants without allergy, infants with allergy in Western societies might be less frequently colonized with infant-type Bifidobacterium species, such as B. longum (biotype longum and infantis), B. bifidum and B. breve, and could be more frequently colonized by B. adolescentis and other species typical of the adult intestinal microbiota (7–10). Therefore, the composition of Bifidobacterium in infants is a prospective target in the management of allergic disease.
Mast cells and basophiles play an essential role in the pathogenesis of allergic reactions such as atopic dermatitis and asthma (11). Mast cells express a high-affinity Fc receptor for IgE and contain numerous mediator-filled granules. Antigen-induced cross-linkage of IgE bound to Fc receptors of mast cells causes the release of their granule content (degranulation) as well as the synthesis and secretion of other mediators, which leads to an immediate allergic reaction. Therefore, when developing anti-allergic pharmaceutical drugs, the emphasis should be on stabilization of mast cells and suppression of degranulation. Recently, some foods such as teas, fruit and vegetables have been proposed for alleviation of symptoms of allergic disease, because certain components of these foods can inhibit the activation of mast cells (12, 13). Furthermore, some non-pathogenic commensal and intestinal mucosa-associated bacteria have been found to function as strong direct inhibitors of mast cell degranulation (14–16).
In the present study, the rat basophilic leukemia mast cell line RBL-2H3 was used to test 16 strains of bifidobacteria isolated from human intestine and a probiotic strain, LGG, for their ability to suppress IgE-mediated degranulation of mast cells.
Sixteen strains of bifidobacteria, originally isolated from human intestine and stored at the Microbiological Laboratory of Takanashi Milk Products (Yokohama, Japan), were used (5). The probiotic strain, LGG (ATCC 53103), was supplied by Valio (Helsinki, Finland). Before use in experiments, bifidobacteria were cultured anaerobically using an Anaero Pack Kenki System (Mitsubishi Gas Chemical, Tokyo, Japan) in GAM broth (Nissui Seiyaku, Tokyo, Japan) at 37°C for 48 hr. Lactobacilli were routinely cultured at 37°C for 18 hr in MRS broth (Becton Dickinson, Sparks, MD, USA). After incubation, the bacteria were washed with sterilized saline, heat-killed at 100°C for 30 min, and lyophilized. Heat-inactivated bacteria were resuspended in tissue culture medium (see below) at a concentration of 100 μg/ml.
The RBL-2H3 cell line was purchased from ATCC (CRL-2256). The cells were cultured in Eagle's MEM (Wako, Osaka, Japan) supplemented with 10% heat-inactivated FBS (Gibco, Eggenstein, Germany), 2 mM L-glutamine, and 100 U/ml of penicillin.
The engagement of high-affinity IgE receptors (FcɛR1) on RBL-2H3 cells, which are known to express mast cell-specific molecules, initiates a number of biochemical events that eventually result in degranulation. The degranulation of RBL-2H3 cells was determined by measuring the release of β-hexosaminidase. The cells (3.0 × 105) were seeded in a 24-well plate and cultured overnight, then washed with PBS and cultured with or without bifidobacteria and lactobacilli (10 μg/ml) for 3 hr at 37°C in an atmosphere of air with 5% CO2. Next, the cells were washed twice with Eagle's MEM and stimulated with monoclonal TNP IgE (clone IgE-3) (40 ng/ml; BD Pharmingen, Tokyo, Japan). After being incubated for 2 hr at 37°C in an atmosphere of air with 5% CO2, the cells were washed once with Tyrode buffer (126 mM NaCl, 5.6 mM glucose, 4.0 mM KCl, 0.6 mM KH2PO4, 10.0 mM HEPES, 0.6 mM MgCl2/6H2O, 1.0 mM CaCl2, and 0.1% BSA; pH 7.4) and then stimulated with TNP-BSA (3 ng/ml) for 1 hr at 37°C. The supernatants were transferred to a 96-well microplate and 0.2% Triton X-100 added to the cells, which were then incubated with 1.3 mg/ml of p-nitrophenyl-N-acetyl-β-D-glucopyranoside (Nakarai, Kyoto, Japan) in 0.1 M sodium citrate buffer (pH 4.5) for 40 min at 37°C. After adding a stop solution (0.2 M glycine; pH 11.0), the optical density at 450 nm was measured using a microplate reader (ImmunoMin NJ-2300; InterMed, Osaka, Japan). The total β-hexosaminidase content was determined by cell lysis with 0.1% Triton-X 100. Inhibition (%) of the release of β-hexosaminidase in the test sample was calculated using the following equation: Inhibition (%) = 100 −[(T/A)/(C/A) × 100]  in which C (control) is antigen-induced β-hexosaminidase release in the absence of Bifidobacterium or Lactobacillus[TNP-BSA(+) − TNP-BSA(−)], T is the antigen-induced β-hexosaminidase of the test sample [TNP-BSA(+) − TNP-BSA(−)], and A is the total β-hexosaminidase content (Triton X-100 extract).
The results were expressed as mean ± SD (n= 3). The statistical significance of differences between the two groups was calculated using the unpaired Student's t-test or Welch's t-test after an F-test.
IgE-mediated degranulation by RBL-2H3 cells was measured by analyzing β-hexosaminidase in the culture supernatant. LGG inhibited IgE-mediated degranulation of RBL-2H3 cells by 50.3%. The 16 tested bifidobacteria suppressed IgE-mediated degranulation by 1.6–57.7%, each bacterium having its own characteristic inhibitory effect on IgE-mediated degranulation of RBL-2H3 (Table 1). Of the tested bifidobacteria, B. bifidum TMC3116 from healthy infants expressed the strongest inhibitory effect (57.7%) on IgE-mediated degranulation of RBL-2H3 cells, whereas B. adolescentis TMC2737 from allergic infants suppressed degranulation by only 13.6%. The bifidobacteria originally isolated from healthy infants suppressed IgE-mediated degranulation more than those isolated from allergic infants, although the difference was not statistically significant (P= 0.25). On the other hand, bacteria taxonomically identified as B. bifidum significantly suppressed IgE-mediated degranulation of RBL-2H3 cells more than those taxonomically identified as B. adolescentis (Fig. 1) (P < 0.05).
Table 1. Rates of inhibition of ß-hexosaminidase release by the bifidobacteria and lactobacillus tested in this study
|Species Name||Strain No.||Origin||Inhibition rate (%)|
|B. adolescentis||TMC 2701||Healthy adult|| 1.62 ± 13.85|
|B. adolescentis||TMC 2704||Healthy adult||20.15 ± 1.77|
|B. adolescentis||TMC 2705||Healthy adult||33.62 ± 3.17|
|B. adolescentis||TMC 2710||Healthy adult||26.80 ± 28.46|
|B. adolescentis||TMC 2718||Allergic infant||31.25 ± 15.29|
|B. adolescentis||TMC 2720||Allergic infant||53.85 ± 3.19|
|B. adolescentis||TMC 2736||Allergic infant||56.43 ± 3.82|
|B. adolescentis||TMC 2737||Allergic infant||13.55 ± 4.53|
|B. adolescentis||TMC 2738||Allergic infant||47.89 ± 6.13|
|B. adolescentis||TMC 2739||Allergic infant||24.60 ± 18.73|
|B. bifidum||TMC 3103||Dairy food||41.77 ± 11.76|
|B. bifidum||TMC 3108||Healthy infant||55.70 ± 3.72|
|B. bifidum||TMC 3110||Healthy infant||42.13 ± 3.53|
|B. bifidum||TMC 3111||Healthy infant||55.71 ± 4.51|
|B. bifidum||TMC 3115||Healthy infant||41.30 ± 16.34|
|B. bifidum||TMC 3116||Healthy infant||57.71 ± 2.59|
|L.rhamnosus||GG||Human intestine||50.34 ± 3.14|
Figure 1. Comparison of inhibitory effect of B. adolescentis and B. bifidum on β-hexosaminidase release. RBL-2H3 cells were preincubated with bifidobacteria for 3 hr prior to IgE sensitization. After stimulation with antigen, degranulation was determined by measuring the release of β-hexosaminidase. The inhibition rates of each of the ten B. adolescentis strains and six B. bifidum strains shown in Table 1 are represented by open circles and the overall inhibition rates for each group are expressed as means ± SD. *, difference between B. adolescentis and B. bifidum group is significant (P < 0.05).
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RBL-2H3 is a well-established cell line used as a model of mast cells and the release of β-hexosaminidase is a well-established indicator of IgE-mediated degranulation. RBL-2H3 cells have been widely used as a test model to evaluate the potent anti-allergic effects of flavonoids, peptides and other food ingredients, and environmental components through their property of suppressing IgE-mediated degranulation of these cells (17). In the present study, the human intestinal bifidobacteria used were examined for their ability to activate mast cells using RBL-2H3 cells. Each of the tested bifidobacteria expressed its characteristic inhibitory effect against IgE-mediated degranulation in RBL-2H3 cells in a strain-dependent manner. Compared with the infant-type species B. bifidum, of all the tested strains which strongly inhibited degranulation the adult-type species B. adolescentis showed the greatest diversity in its ability to affect IgE-mediated degranulation. B. adolescentis strains such as TMC2701 and TMC2704 from healthy adults and TMC2737 from allergic infants showed relatively reduced ability to suppress degranulation compared with strains of B. bifidum. These results are in good agreement with those of our previous study in which strains of B. adolescentis differed from those of B. bifidum with resect to their immune modulatory effects (5, 18). To our knowledge, the present study is the first to indicate that different bifidobacteria influence IgE-mediated degranulation of mast cells differently, infant-type bifidobacteria such as B. bifidum having a greater influence on IgE-mediated allergic inflammation than other species such as B. adolescentis.
LGG has been shown to reduce the clinical symptoms of atopic eczema in breast-feeding infants whose mothers are given LGG, and in milk-hypersensitive infants who consume a hydrolyzed whey formula supplemented with LGG. A 7-year clinical follow-up study has shown that this probiotic bacterium is effective in protecting infants with a genetically high risk of allergic disease from the development of atopic disease (19, 20). In other well-designed clinical studies, LGG improved atopic eczema/dermatitis, stimulation of interferon-γ production being suggested as one of the possible mechanisms for this bacterium's health-promoting effects (21, 22). In another recent study, fermented milk prepared with LGG was found to improve the allergic symptoms of Japanese cedar pollinosis (23). However, LGG did not modulate serum IgE synthesis in these allergic subjects, although it has been found to slightly affect serum IgE concentrations in ovalbumin-sensitized mice (24). On other hand, in NC/Nga mice, a model of human atopic dermatitis, ingestion of heat-treated LGG inhibited the onset and development of atopic skin lesions, and this effect was accompanied by reduced degranulation and infiltration of mast cells and eosinophils (25). These results suggest that another underlying mechanism by which LGG exerts its health-promoting effects in allergic patients could be via a direct influence on mast cells. In the present study, the observation that some bifidobacteria from healthy infants affect mast cells in the same way as LGG does suggests that these bifidobacteria possess similar health promoting effects in allergic subjects as those observed in LGG related studies.
In order to clarify the underlying mechanisms by which tested bacteria expressed inhibitory effects against IgE-mediated degranulation of mast cells, LGG was tested for its abilities to influence IgE binding to FcɛRI, activate expression of an intracellular signaling cascade, and stimulate secretion of cytokines such as TNF-α from RBL-2H3 cells. Some slight effects were observed, leaving the mechanisms by which LGG affects mast cells unclear.