Inhibition by troglitazone of the antigen-induced production of leukotrienes in immunoglobulin E-sensitized RBL-2H3 cells

Abstract

1. The effect of troglitazone, an anti-diabetic drug with insulin-sensitizing action, on antigen-induced production of leukotriene (LT) B(4), C(4) and E(4) and prostaglandin D(2) (PGD(2)) was examined in dinitrophenol (DNP)-specific immunoglobulin E (IgE)-sensitized RBL-2H3 mast cells following stimulation by the antigen, DNP-conjugated human serum albumin. Levels of LTB(4), C(4) and E(4) and PGD(2) in the conditioned medium were enzyme-immunoassayed. 2. Troglitazone inhibited the antigen-induced production of LTB(4), C(4) and E(4) and the potency of the inhibition was comparable to that of zileuton, a specific inhibitor of 5-lipoxygenase (5-LOX) and a clinically used anti-asthmatic drug. Neither troglitazone nor zileuton affected antigen-induced production of PGD(2), arachidonic acid release from membrane phospholipids and degranulation. 3. Troglitazone inhibited LTB(4) production by the supernatant fraction of RBL-2H3 cell lysate with similar potency to zileuton, suggesting that troglitazone inhibits LT production by direct inhibition of 5-LOX activity. 4. Furthermore, it was shown that troglitazone as well as zileuton inhibited LTB(4) production in A23187-stimulated rat peritoneal neutrophils. 5. These findings suggest that troglitazone inhibits antigen-induced LT production in the IgE-sensitized RBL-2H3 cells and A23187-stimulated rat peritoneal neutrophils by direct inhibition of 5-LOX activity.

Figure 1

Effects of the antigen DNP-HSA on the production of LTB₄, C₄, E₄ and PGD₂ by RBL-2H3 cells. (a) RBL-2H3 cells (5×10⁵ cells) were incubated for 15 min at 37°C in 1 ml of PIPES buffer containing the indicated concentrations of DNP-HSA. (b) RBL-2H3 cells (5×10⁵ cells) were incubated for 0, 5, 15, 30 and 60 min at 37°C in 1 ml of PIPES buffer containing DNP-HSA (50 ng ml⁻¹). Values are the means±s.e.mean from four wells; each s.e.mean is within the symbol. Without the antigen stimulation, levels of each eicosanoid in the conditioned medium were less than the detectable amount.

Figure 2

Effects of preincubation time with troglitazone on the antigen-induced LTC₄ production in RBL-2H3 cells. RBL-2H3 cells (5×10⁵ cells) were preincubated for the periods indicated at 37°C in 1 ml of medium containing 1 μM of troglitazone. After three washes, the cells were further incubated for 30 min at 37°C in 1 ml of PIPES buffer containing DNP-HSA (50 ng ml⁻¹) and 1 μM of troglitazone. Values are the means±s.e.mean from four wells. Statistical significance: ^***P<0.001 vs the group without preincubation (0 min).

Figure 3

Effects of troglitazone and zileuton on the production of LTB₄ by 20,000×g supernatant fraction of intact RBL-2H3 cells. RBL-2H3 cells (2.5×10⁷ cells) were lysed by sonication, and the lysate was centrifuged at 20,000×g and 4°C for 20 min. An aliquot of the supernatant fraction was preincubated for 2 h at 37°C in the presence of the indicated concentrations of troglitazone or zileuton, and further incubated for 30 min at 37°C in the presence of arachidonic acid (1 μM). LTB₄ produced is expressed as ng LTB₄ per 1 μg protein. Values are the means±s.e.mean from four samples. Statistical significance: ^*P<0.01, ^***P<0.001 vs the control.

Figure 4

Effects of troglitazone and zileuton on LTB₄ production in rat peritoneal neutrophils. Rat peritoneal neutrophils (1×10⁶ cells) were preincubated for 2 h at 37°C in 1 ml of medium containing troglitazone or zileuton. After three washes, the cells were incubated for 30 min at 37°C in 1 ml of PIPES buffer containing A23187 (1 μM) and the corresponding concentrations of troglitazone or zileuton. Values are the means±s.e.mean from four wells. Statistical significance: ^**P<0.01, ^***P<0.001 vs the control.