Leukotriene B 4 level in neutrophils from allergic and healthy subjects stimulated by low concentration of calcium ionophore A23187. Effect of exogenous arachidonic acid and possible endogenous source

Abstract

Peripheral blood neutrophils from patients with allergic rhinitis and from normal subjects were incubated for 5 min at 37°C with 0.15 μM calcium ionophore A23187 in the absence or presence of exogenous arachidonic acid (2.5 to 10 μM). In neutrophils from allergic patients, the leukotriene B 4 (LTB 4) level was significantly increased by exogenous arachidonic acid in a concentration-dependent manner (16.2 ± 4.2 and 38.1 ± 6.8 pmol/5 min per 2·10 6 cells in the absence and presence of 10 μM arachidonic acid, respectively; P < 0.005; n = 8). The LTB 4 level in neutrophils from healthy subjects was only 0.97 ± 0.17 pmol/5 in per 2·10 6 cells ( n = 5) and was not enhanced by exogenous arachidonate. When cells from allergic patients were challenged in the presence of exogenous [1- 14C]arachidonic acid, released LTB 4 was radiolabeled and the incorporated radioactivity increased with the labeled arachidonate concentration. Labeled LTB 4 was never detectable after incubating neutrophils from normal donors with exogenous labeled arachidonate. When neutrophils were incubated with [1- 14C]arachidonate for 1 h, the different lipid pools of the two cell populations were labeled but both types of neutrophils produced unlabeled LTB 4 in response to ionophore stimulation. The hydrolysis of choline and ethanolamine phospholipids into diacyl-, alkenylacyl- and alkylacyl-species revealed that solely the alkylacyl-subclass of phosphatidylcholine was unlabeled. We conclude (i) that neutrophils from allergic patients stimulated by low ionophore concentration produce more LTB 4 than neutrophils from healthy subjects and incorporate exogenous arachidonate, (ii) that endogenous arachidonate converted to LTB 4 by the 5-lipoxygenase pathway may provide only from 1- O-alkyl-2-arachidonoyl-glycero-3-phosphocholine.