THE INVOLVEMENT OF PHOSPHATIDYLINOSITOL 3-KINASE IN CRYSTAL-INDUCED HUMAN NEUTROPHIL ACTIVATION

Objective, We investigated whether phosphatidylinositol (PI) 3-kinase is involved in the signal transduction pathway leading to neutrophil a ctivation by inflammatory microcrystals. Methods, Neutrophil chemilumi nescence and degranulation responses to opsonized crystals were measur ed in the presence of selective inhibitors known to inhibit PI 3-kinas e activity in neutrophils. Results, Wortmannin and LY 294002, 2 select ive inhibitors of PI 3-kinase, were shown to inhibit neutrophil activa tion induced by plasma opsonized crystals of calcium pyrophosphate dih ydrate (CPPD) [both monoclinic (M) and triclinic (T) forms] and monoso dium urate monohydrate (MSUM). IC50 for wortmannin or LY 294002 inhibi tion of crystal induced respiratory burst (measured by chemiluminescen ce) was about 3 nM and 0.3 mu M, respectively, proving the pivotal rol e of PI 3-kinase in neutrophil respiratory burst activation by all 3 c rystals. 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-k inase in signal transduction pathways leading to crystal induced neutr ophil degranulation. All 3 crystals induced the activation of PI 3-kin ase in neutrophils as measured by the increased PI 3-kinase activity a ssociated with immunoprecipitated tyrosine phosphorylated proteins fro m I min crystal-neutrophil incubations. Neutrophils pretreated with wo rtmannin at 10 nM showed sub-basal levels of PI 3-kinase activity at a ll time points measured. Conclusion. PI3-kinase plays a central role i n the signal transduction pathways leading to respiratory burst and de granulation responses in neutrophils activated by inflammatory microcr ystals.