CRYSTAL-INDUCED NEUTROPHIL ACTIVATION .3. INFLAMMATORY MICROCRYSTALS INDUCE A DISTINCT PATTERN OF TYROSINE PHOSPHORYLATION IN HUMAN NEUTROPHILS

The activation of human neutrophils by monosodium urate and calcium pyrophosphate dihydrate crystals is believed to play a critical role in the pathogenesis of arthritides such as acute gout and pseudogout, respectively. In this study, we investigated the potential involvement of tyrosine phosphorylation in microcrystal-mediated activation of human neutrophils. Immunoblot analysis with antiphosphotyrosine antibodies demonstrated that triclinic monosodium urate and calcium pyrophosphate dihydrate crystals stimulated a time- and concentration-dependent tyrosine phosphorylation of at least five proteins (pp130, 118,80, 70, and 60). While phosphoprotein (pp) 118 and pp70 were the major phosphorylated substrates, pp70 was the dominant one in reactivity with antiphosphotyrosine antibodies. When the temporal patterns, as well as the levels of tyrosine phosphorylation for both types of crystals were compared, monosodium urate crystals were found to be more potent activators than calcium pyrophosphate dihydrate crystals. The tyrosine phosphorylation patterns induced by microcrystals differed from those stimulated by other soluble (FMLP, C5a, or leukotriene B4) or particulate (unopsonized latex beads or zymosan) agonists which stimulated preferentially the tyrosine phosphorylation of pp118. The ratio of the intensities of pp118 and pp70 were specific of the stimulation with microcrystals when compared to those observed with the other soluble or particulate agonists. Colchicine, a drug used specifically in the treatment of gout and pseudogout, inhibited microcrystal-induced tyrosine phosphorylation, while beta- and gamma-lumicolchicine were without effect. On the other hand, colchicine failed to inhibit FMLP-induced tyrosine phosphorylation. Furthermore, while colchicine inhibited the activation of the NADPH oxidase by microcrystals, it, on the other hand, enhanced the production of superoxide anions by FMLP. Taken together, these results (a) demonstrate that tyrosine phosphorylation is involved in the mechanism of activation of human neutrophils induced by microcrystals; and (b) suggest, on the basis of the characteristics of the observed patterns of tyrosine phosphorylation, that this response may be specific to the microcrystals and relevant to their phlogistic properties.