The involvement of phosphatidylinositol 3-kinase in crystal induced human neutrophil activation.

Abstract

OBJECTIVE: We investigated whether phosphatidylinositol (PI) 3-kinase is involved in the signal transduction pathway leading to neutrophil activation by inflammatory microcrystals. METHODS: Neutrophil chemiluminescence and degranulation responses to opsonized crystals were measured in the presence of selective inhibitors known to inhibit PI 3-kinase activity in neutrophils. RESULTS: Wortmannin and LY 294002, 2 selective inhibitors of PI 3-kinase, were shown to inhibit neutrophil activation induced by plasma opsonized crystals of calcium pyrophosphate dihydrate (CPPD) [both monoclinic (M) and triclinic (T) forms] and monosodium urate monohydrate (MSUM). IC50 for wortmannin or LY 294002 inhibition of crystal induced respiratory burst (measured by chemiluminescence) was about 3 nM and 0.3 microM, respectively, proving the pivotal role of PI 3-kinase in neutrophil respiratory burst activation by all 3 crystals. Degranulation responses of neutrophils to CPPD(M) and CPPD(T) crystals were also inhibited by about 50% by wortmannin in the 10 to 20 nM concentration range, supporting the direct involvement of PI 3-kinase in signal transduction pathways leading to crystal induced neutrophil degranulation. All 3 crystals induced the activation of PI 3-kinase in neutrophils as measured by the increased PI 3-kinase activity associated with immunoprecipitated tyrosine phosphorylated proteins from 1 min crystal-neutrophil incubations. Neutrophils pretreated with wortmannin at 10 nM showed sub-basal levels of PI 3-kinase activity at all time points measured. CONCLUSION: PI 3-kinase plays a central role in the signal transduction pathways leading to respiratory burst and degranulation responses in neutrophils activated by inflammatory microcrystals.