Requirement of Phosphatidylinositol 3-Kinase Activation and Calcium Influx for Leukotriene B4-induced Enzyme Release

Abstract

Leukotriene B 4 (LTB 4 ) is a potent lipid mediator involved in host defense and inflammatory responses. It causes chemotaxis, generation of reactive oxygen species, and degranulation. However, only little is known of the molecular mechanisms by which LTB 4 induces these biological activities. To analyze the intracellular signaling pathways to mediate lysosomal enzyme release through the cloned LTB 4 receptor (BLT1), we transfected BLT1 to rat basophilic leukemia cells (RBL-2H3). LTB 4 dose-dependently released ␤-hexosaminidase, and the release was mostly inhibited when the cells were pretreated with pertussis toxin, indicating that the degranulation is mediated by G i proteins. LTB 4 activated phosphatidylinositol 3-kinase (PI3-K) through G i , and inhibition of PI3-K by wortmannin or LY290042 inhibited degranulation. Granulocytes from PI3-K␥-deficient mice showed reduced LTB 4 -induced degranulation, suggesting that this isozyme of PI3-K is involved in the degranulation. LTB 4 also caused calcium release from intracellular stores and calcium influx from the outside milieu through G i , but only the calcium influx is critical for the lysosomal enzyme release. Calcium influx and PI3-K activation are both downstream events of G i , since they were inhibited by pertussis toxin. These two events are in essence independent each other, because calcium depletion did not affect PI3-K, and inhibition of PI3-K did not attenuate calcium influx significantly. Thus, our results have clearly shown that LTB 4 binds BLT1 and activates G i -like protein, and both PI3-K␥ activation and a sustained calcium elevation by calcium influx are necessary for enzyme release in these cells.Leukotreine B 4 (LTB 4 ), 1 a metabolite of arachidonic acid, is one of the most potent chemoattractants for neutrophils. LTB 4 activates leukocytes, leading to chemotaxis, release of lysosomal enzyme, and production of superoxide anions, thus playing important roles in host defense (1, 2). Overproduction of LTB 4 is involved in various inflammatory diseases including psoriasis (3), bronchial asthma (4), rheumatoid arthritis (5), inflammatory bowel diseases (6), and ischemic renal failure (7). LTB 4 binds to a specific G protein-coupled receptor named BLT1, which was cloned in our laboratory (8). Signaling to activate leukocytes through BLT1 is poorly understood, in part because most of the previous studies have been done using peripheral leukocytes, especially granulocytes. The lifetime of these cells is short (several days), which makes it difficult to perform studies using various drugs or gene transfection. We have previously analyzed signaling pathway from BLT1 using Chinese hamster ovary cells stably expressing human BLT1 (8). In these cells, LTB 4 increases intracellular IP3 levels by G q -like protein(s), inhibits adenylyl cyclase activity, and activates chemotaxis through G i -like protein(s). In hematopoietic cells, however, the intracellular events leading to release of lysosomal enzymes and generation of oxygen species are poorly understood....

Leukotriene B4 Induces Release of Antimicrobial Peptides in Lungs of Virally Infected Mice

“…This hypothesis is now under investigation. Moreover, because BLT1-mediated neutrophil degranulation involves the activation of the Src family kinase member Yes (59) and PI3K (60), it would be of great interest to further study the intracellular events implicated in BLT1-mediated antiviral neutrophil granule secretion.…”

Involvement of BLT1 Endocytosis and Yes Kinase Activation in Leukotriene B4-Induced Neutrophil Degranulation

“…PLB-985 cells were also used by Kaldi et al (52) to show that fMLP-mediated degranulation was dependent on the presence of cholesterol in the cell membrane. Because the only report on BLT1-mediated signaling events involved in degranulation was studied using rat RBL-2H3 cells stably transfected with BLT1 cDNA (29), it was of great interest to use a human cellular model such as PLB-985 cells to study human neutrophil degranulation.…”

“…BLT1-mediated degranulation in RBL-2H3 cells stably transfected with BLT1 cDNA was shown to be partially dependent on PI3K activity (29). To verify whether this was the case in our PLB-BLT model, the specific PI3K inhibitors LY294002 (10 M) and wortmannin (1 M) were used.…”

“…Many stimuli can lead to neutrophil degranulation including LTB 4 (28). At this time, the only characterized BLT1-dependent intracellular signaling events leading to degranulation have been reported using the RBL-2H3 rat basophilic leukemia cell line stably transfected with wild-type BLT1 cDNA (29). These intracellular signaling events include calcium influx and PI3K activation.…”

Increased Acute Inflammation, Leukotriene B4-Induced Chemotaxis, and Signaling in Mice Deficient for G Protein-Coupled Receptor Kinase 6

“…The rapid and transient LTB 4 -induced increase in intracellular calcium is a major player in BLT1 signaling (29). To investigate whether BLT1 signaling is changed in the absence of GRK6, we investigated LTB 4 -induced calcium responses in WT and GRK6 Ϫ/Ϫ PMN.…”