Effect of Gal-9 on intracellular Ca²⁺ mobilization. A, Fluo-3-AM-loaded RBL-2H3 cells were sequentially added with 1 μm sGal-9 (or PBS for control), 0.1 μg/ml Spe7, and then 0.016 μg/ml DNP-HSA at 30-min intervals as indicated by the arrows. The assay was carried out in the absence (upper and middle) or presence (lower) of 20 mm lactose. The fluorescence signal is expressed on the vertical axes as relative fluorescence change (F/F₀) from the initial signal value (Mean ± S.D., n = 3). B, no relations between Gal-9-dependent Ca²⁺ mobilization and the anti-degranulation effect of Gal-9 are shown. The assay procedure was basically the same as in Fig. 2A, except that 20 mm lactose or control buffer was added 10 min after the addition of 1 μm Gal-9, and then the cells were stimulated with Spe7 and DNP-HSA for β-HEX release (mean ± S.D., n = 3). Representative data of twice reproduced are shown.

Expression and release of Gal-9 from mast cells. A, shown is ELISA analysis of Gal-9 expression in T-cell lines (Jurkat and Molt-4) and mast cell lines (HMC-1 and MC/9). B, release of Gal-9 from mast cells by the degranulation stimuli is shown. Mouse mast cell line MC/9 was incubated with the indicated concentrations of Spe7 and DNP-HSA for 30 min or 48 h to measure the released LTC₄ (left) or Gal-9 (right), respectively, using specific ELISA. After 48 h incubation, cell number was 5–30% higher in the presence of Spe7 and DNP-HSA compared with the control. Therefore, the amount of Gal-9 in the culture supernatant was compensated by the cell number and expressed on the vertical axis as release of Gal-9 per 10⁶ cells. Data are representative of at least two experiments (mean ± S.D., n = 3).

In vivo effects of Gal-9 in asthma and allergy models. A, to induce asthmatic reactions, guinea pigs were sensitized by inhaled 1% OVA for 8 days and challenged with 2% OVA to induce asthmatic reactions, and then sRaw was determined at the indicated time points. sGal-9 was administrated intraperitoneally (1 mg/body) 30 min before the OVA treatments in both the sensitization and challenge. The data are expressed by percent changes in sRaw. The amount of total and OVA-specific IgE and IgG₁ was measured by ELISA using serum obtained after the last sRaw measurement. Data shown are the mean ± S.E., n = 7 or 8; **, p < 0.01 compared with PBS control. B, effect on passive cutaneous anaphylaxis in mice. Ear edema was induced in mice by topical challenge with 2,4-dinitrofluorobenzene (DNFB) on the right ear of Spe7-sensitized mice. sGal-9 (left) or ketotifen (right) was administered intraperitoneally 30 min before the challenge. The thickness of the ears was measured and is expressed on the vertical axes as ear swelling (thickness of the right ear over the left; mean ± S.E., n = 4 or 5). ***, p < 0.001 compared with PBS control.

Attenuation of mediator release from rat basophilic leukemia RBL-2H3 cells by Gal-9. A, shown is the effect on β-HEX release by sGal-9 and natural-form Gal-9. RBL-2H3 cells were incubated with the indicated concentration of Gal-9 for 10 min in the presence or absence of 20 mm lactose or sucrose. Then the cells were sequentially added and incubated with Spe7 (0.1 μg/ml) for 30 min followed by DNP-HSA (0.016 μg/ml). The supernatants were collected 60 min after the stimulation for β-HEX assay, and the amount of the enzyme released into the supernatants was expressed as the percentage of total β-HEX in the cells. w/o, without. B and C, shown is the effect on histamine and LTC₄ release. RBL-2H3 cells were stimulated as above in the presence or absence of 1 μm Gal-9 or sGal-9. The supernatants were collected 5 and 20 min after the stimulation for histamine and LTC₄ assays, respectively. D, shown is suppression of β-HEX release from IgE-sensitized RBL-2H3 cells. Cells were stimulated as described in A, whereas sGal-9 was added 10 min before Spe7, 30 min after Spe7, or 30 min after the addition of DNP-HSA. E, the experimental procedure is basically the same to as in D, but RBL-2H3 cells were stimulated by rat IgE IR162 (1 μg/ml) and anti-rat IgE antibody MARE1 (0.89 μg/ml). All the data are derived from a single representative experiment of more than two and are shown as the mean ± S.D. (n = 3).

Binding of Gal-9 to IgE prevent IgE-antigen complex formation. A, human IgG₁ from myeloid, human IgE from myeloid, mouse monoclonal IgE Spe7, and rat monoclonal IgE IR162 were coated in 96-well plates and allowed to interact with biotinylated-galectins. Relative binding is expressed on the vertical axes in comparison with the binding of Gal-1 (mean ± S.D., n = 3). B, prevention of IgE-antigen complex formation by Gal-9 in 96-well plates is shown. DNP-HSA-coated 96-well plates were incubated with 0.1 μg/ml biotinylated Spe7 for 60 min in the indicated concentration of galectins in the presence (right) or absence (left) of 20 mm lactose. The data are expressed by the percentage of Spe7 binding as the binding without galectins is 100% (mean ± S.D., n = 3). C, no effect of Gal-9 on periodate-treated IgE is shown. Biotinylated Spe7 was subjected to periodate oxidation to disrupt carbohydrate chains. The samples were sieved by SDS-PAGE and silver-stained (left) and used for binding assay against DNP-HSA in the presence or absence of 1 μm sGal-9 and 20 mm lactose as indicated (right). The assay condition is basically the same as B. Periodate- and mock-treated Spe7 were used without compensating for the loss of protein during the treatment, and the binding of mock-treated Spe7 in the absence of sGal-9 and lactose was set as 100% (mean ± S.D., n = 4). D, RBL-2H3 cells pre-bound with 0.1 μg/ml biotinylated Spe7 were incubated with 1 μm sGal-9 when indicated, and Spe7, which stayed on the cell surface, was detected by labeling the cells with streptavidin-conjugated phycoerythrin and FACS analysis. The mean fluorescence values are shown in the bar chart (mean ± S.D., n = 3). Dark gray histogram, Spe7⁻sGal-9⁻; pale gray histogram, Spe7⁺sGal-9⁻; open histogram, Spe7⁺sGal-9⁺. E, RBL-2H3 cells pre-bound with 0.1 μg/ml Spe7 (left) or 1 μg/ml IR162 (right) were incubated with either 0.016 μg/ml DNP-conjugated biotinylated BSA or 0.89 μg/ml biotinylated-MARE1 in the presence or absence of 1 μm sGal-9 as indicated. The binding of DNP-BSA or MARE1 to the cells was determined by labeling the cells with streptavidin-conjugated phycoerythrin and FACS analysis. The mean fluorescent values are summarized in the bar chart (mean ± S.D., n = 3). Dark gray histogram, Spe7⁻sGal-9⁻DNP⁻ or IR162⁻sGal-9⁻MARE1⁻; pale gray histogram, Spe7⁺sGal-9⁻DNP⁺ or IR162⁺sGal-9⁻MARE1⁺; open histogram, Spe7⁺sGal-9⁺DNP⁺ or IR162⁺sGal-9⁺MARE1⁺. Representative data of at least twice-reproduced experiments are shown.