A new method to evaluate anti-allergic effect of food component by measuring leukotriene B4 from a mouse mast cell line

Abstract

Leukotrienes (LTs), chemical mediators produced by mast cells, play an important role in allergic symptoms such as food allergies and hay fever. We tried to construct an evaluation method for the anti-LTB₄ activity of chemical substances using a mast cell line, PB-3c. PB-3c pre-cultured with or without arachidonic acid (AA) was stimulated by calcium ionophore (A23187) for 20 min, and LTB₄ production by the cells was determined by HPLC with UV detection. LTB₄ was not detected when PB-3c was pre-cultured without AA. On the other hand, LTB₄ production by PB-3c pre-cultured with AA was detectable by HPLC, and the optimal conditions of PB-3c for LTB₄ detection were to utilize the cells pre-cultured with 50 µM AA for 48 h. MK-886 (5-lipoxygenase inhibitor) completely inhibited LTB₄ production, but AACOCF₃ (phospholipase A₂ inhibitor) slightly increased LTB₄ production, suggesting that LTB₄ was generated from exogenous free AA through 5-lipoxygenase pathway. We applied this technique to the evaluation of the anti-LTB₄ activity of food components. PB-3c pre-cultured with 50 µM AA for 48 h was stimulated with A23187 in the presence of 50 µM soybean isoflavones (daidzin, genistin, daidzein, and genistein), equol, quercetin, or kaempferol. Genistein, equol, quercetin, and kaempferol strongly inhibited LTB₄ production without cytotoxicity. These results suggest that a new assay system using PB-3c is convenient to evaluate LTB₄ inhibition activity by food components. This method could be utilized for elucidation of the mechanisms of LTB₄ release suppression by food components such as flavonoids and the structure-activity relationship.

Keywords: Allergy; Flavonoid; Isoflavone; Leukotriene; Mast cells.

Fig. 1

Structures of soybean isoflavones and flavonols

Fig. 2

Effect of arachidonic acid (AA) concentration during pre-culture of PB-3c on LTB₄ production. A mast cell line, PB-3c (2 × 10⁶ cells) was pre-cultured in RPMI-1640 containing 0–100 µM of AA for 48 h. The cells were stimulated with 5 µM calcium ionophore (A23187) for 20 min and then LTB₄ produced by the cells was determined by HPLC with UV detection. Data represent mean ± SE (n = 4). Means without a common letter are significantly different (p < 0.01). N.D. not detected

Fig. 3

Effect of pre-culture period of PB-3c with AA before the stimulation on LTB₄ production. PB-3c (2 × 10⁶ cells) was pre-cultured in RPMI-1640 containing 50 µM AA for 0–72 h. The cells were stimulated with 5 µM A23187 for 20 min and then LTB₄ produced by the cells was determined by HPLC with UV detection. Data represent mean ± SE (n = 4)

Fig. 4

Effect of enzyme inhibitors related to arachidonate cascade on LTB₄ production by PB-3c. PB-3c was pre-cultured with 50 µM AA for 48 h and then stimulated with 5 µM A23187 in the presence of MK-886 (5-LOX inhibitor) and AACOCF₃ (phospholipase A₂ inhibitor). LTB₄ was determined by HPLC with UV detection. Data represent mean ± SE (n = 4). Means without a common letter are significantly different (p < 0.01). N.D. not detected

Fig. 5

Effect of soybean isoflavones and equol on LTB₄ production by PB-3c. PB-3c was pre-cultured with 50 µM AA for 48 h and then stimulated with 5 µM A23187 in the presence of 50 µM daidzin, genistin, daidzein, genistein, and equol. LTB₄ was determined by HPLC with UV detection. Data represent mean ± SE (n = 4). Means without a common letter are significantly different (p < 0.01). N.D. not detected

Fig. 6

Effect of flavonoids on LTB₄ production by PB-3c. PB-3c was pre-cultured with 50 µM AA for 48 h and then stimulated with 5 µM A23187 in the presence of 50 µM quercetin and kaempferol. LTB₄ was determined by HPLC with UV detection. Data represent mean ± SE (n = 4). Means without a common letter are significantly different (p < 0.01). N.D. not detected