BACKGROUND: Allergic disease is the result of an interplay of many different cell types, including basophils and mast cells, in combination with various inflammatory lipid mediators, such as platelet-activating factor (PAF) and leukotrienes (LT). LTC4 synthesis by human basophils has been studied quite extensively. However, not much is known about the synthesis of PAF by human basophils. OBJECTIVE: In this study, we have made a comprehensive comparison between the kinetics of PAF and LTC4 synthesis, in highly purified basophils, activated with different stimuli or with combinations of stimuli. METHODS: Synthesis of PAF and LTC4 by human basophils was determined with commercially available assay kits. The basophils were activated with C5a, fMLP, PMA, allergen or anti-IgE, in the absence and presence of IL-3 and/or in combination with elevation of cytosolic free Ca2+ by the sarcoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin. RESULTS: Most stimuli were found to induce both PAF and LTC4 synthesis. PAF synthesis and LTC4 release were enhanced by preincubation of the basophils with IL-3 or by elevation of cytosolic free Ca2+ by thapsigargin. Incubation of human basophils with IL-3 alone or thapsigargin alone did not result in detectable synthesis of PAF and LTC4, whereas the combination of the two resulted in high amounts of PAF and LTC4 synthesis. Depending on the stimulus used, LTC4 release was 5-100-fold higher than PAF synthesis. In addition, PAF, but not LTC4, was transiently detected, probably due to PAF degradation. LTC4 and PAF synthesis was strongly blocked by inhibitors of cytosolic phospholipase A2, indicating that this enzyme is involved in PAF and LTC4 synthesis by activated human basophils. CONCLUSION: This study provides a first comprehensive comparison of PAF and LTC4 synthesis in highly purified human basophils, stimulated with a variety of stimuli.
BACKGROUND:Allergic disease is the result of an interplay of many different cell types, including basophils and mast cells, in combination with various inflammatory lipid mediators, such as platelet-activating factor (PAF) and leukotrienes (LT). LTC4 synthesis by human basophils has been studied quite extensively. However, not much is known about the synthesis of PAF by human basophils. OBJECTIVE: In this study, we have made a comprehensive comparison between the kinetics of PAF and LTC4 synthesis, in highly purified basophils, activated with different stimuli or with combinations of stimuli. METHODS: Synthesis of PAF and LTC4 by human basophils was determined with commercially available assay kits. The basophils were activated with C5a, fMLP, PMA, allergen or anti-IgE, in the absence and presence of IL-3 and/or in combination with elevation of cytosolic free Ca2+ by the sarcoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin. RESULTS: Most stimuli were found to induce both PAF and LTC4 synthesis. PAF synthesis and LTC4 release were enhanced by preincubation of the basophils with IL-3 or by elevation of cytosolic free Ca2+ by thapsigargin. Incubation of human basophils with IL-3 alone or thapsigargin alone did not result in detectable synthesis of PAF and LTC4, whereas the combination of the two resulted in high amounts of PAF and LTC4 synthesis. Depending on the stimulus used, LTC4 release was 5-100-fold higher than PAF synthesis. In addition, PAF, but not LTC4, was transiently detected, probably due to PAF degradation. LTC4 and PAF synthesis was strongly blocked by inhibitors of cytosolic phospholipase A2, indicating that this enzyme is involved in PAF and LTC4 synthesis by activated human basophils. CONCLUSION: This study provides a first comprehensive comparison of PAF and LTC4 synthesis in highly purified human basophils, stimulated with a variety of stimuli.
Authors: Barry B Rubin; Gregory P Downey; Adeline Koh; Norbert Degousee; Farideh Ghomashchi; Laxman Nallan; Eva Stefanski; Denis W Harkin; Chunxiang Sun; Brian P Smart; Thomas F Lindsay; Vera Cherepanov; Eric Vachon; David Kelvin; Martin Sadilek; Glenn E Brown; Michael B Yaffe; Jonathan Plumb; Sergio Grinstein; Michael Glogauer; Michael H Gelb Journal: J Biol Chem Date: 2004-10-08 Impact factor: 5.157