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 py rophosphate dihydrate crystals is believed to play a critical role in the pathogenesis of arthritides such as acute gout and pseudogout, res pectively. 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 tyr osine phosphorylation of at least five proteins (pp130, 118,80, 70, an d 60). While phosphoprotein (pp) 118 and pp70 were the major phosphory lated substrates, pp70 was the dominant one in reactivity with antipho sphotyrosine antibodies. When the temporal patterns, as well as the le vels of tyrosine phosphorylation for both types of crystals were compa red, monosodium urate crystals were found to be more potent activators than calcium pyrophosphate dihydrate crystals. The tyrosine phosphory lation patterns induced by microcrystals differed from those stimulate d by other soluble (FMLP, C5a, or leukotriene B4) or particulate (unop sonized latex beads or zymosan) agonists which stimulated preferential ly 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 go ut and pseudogout, inhibited microcrystal-induced tyrosine phosphoryla tion, while beta- and gamma-lumicolchicine were without effect. On the other hand, colchicine failed to inhibit FMLP-induced tyrosine phosph orylation. Furthermore, while colchicine inhibited the activation of t he NADPH oxidase by microcrystals, it, on the other hand, enhanced the production of superoxide anions by FMLP. Taken together, these result s (a) demonstrate that tyrosine phosphorylation is involved in the mec hanism of activation of human neutrophils induced by microcrystals; an d (b) suggest, on the basis of the characteristics of the observed pat terns of tyrosine phosphorylation, that this response may be specific to the microcrystals and relevant to their phlogistic properties.