Crystal-induced Neutrophil Activation IV. Specific Inhibition of Tyrosine Phosphorylation by Colchicine Charles J. Roberge, Murielle Gaudry, Rinaldo de M6dicis, * Andre Lussier, * Patrice E. Poubelle, and Paul H. Naccache Centre de Recherche en Inflammation, Immunologie et Rhumatologie, Centre de Recherche du CHUL and Department of Medicine, Faculty ofMedicine, Universite' Laval, Quebec, Canada; and *Unite des Maladies Rhumatismales, Centre Hospitalier Universitaire, Universite de Sherbrooke, Canada Abstract We recently demonstrated that pathologically relevant inflam- matory microcrystals, namely triclinic monosodium urate (MSU) and calcium pyrophosphate dihydrate (CPPD) crys- tals, potently stimulate a characteristic protein tyrosine phos- phorylation pattern in human neutrophils that differed from that observed in response to other soluble or particulate ago- nists. In this study, the effects of colchicine on protein tyrosine phosphorylation induced by MSU and CPPD crystals in hu- man blood neutrophils were investigated. Immunoblot analysis with antiphosphotyrosine antibodies demonstrated that colchi- cine dose-dependently inhibited the tyrosine phosphorylation of all the proteins phosphorylated in response to MSU and CPPD crystals. Other microtubule-disruptive agents such as vinblastine, nocodazole, and colcemid also inhibited crystal-in- duced protein tyrosine phosphorylation while lumicolchicine and trimethylcolchicinic acid were without effect. Indometha- cin and phenylbutazone were similarly without effect on micro- crystal-induced tyrosine phosphorylation. Colchicine, as well as the other active alkaloids, failed to inhibit the protein tyro- sine phosphorylation elicited by FMLP, C5a, leukotriene B4, and unopsonized zymosan. Overall, these results demonstrate that colchicine specifically and significantly inhibits the protein tyrosine phosphorylation induced by MSU and CPPD crystals and suggest that its effects are associated, at least in part, with its interaction with microtubules. Furthermore, the use of mi- crotubule-disrupting drugs demonstrate that the mechanisms implicated in the induction of protein tyrosine phosphorylation by microcrystals differed from those involved in response to other soluble or particulate agonists. (J. Clin. Invest. 1993. 92:1722-1729.) Key words: arthritis * inflammation * gout - pseudogout - leukocytes - microtubules Introduction The deposition of several distinct microcrystals has been asso- ciated with the pathogenesis of acute and chronic articular syn- dromes. For example, it is now well established that the pres- ence of monosodium urate (MSU)' and calcium pyrophos- Address correspondence to Paul H. Naccache, Ph.D., Centre de Re- cherche-Inflammation, Immunologie et Rhumatologie, Le Centre Hos- pitalier de l'Universite Laval, Quebec G 1 V 4G2, Canada. Receivedfor publication 26 February 1993 and in revisedform 6 May 1993. phate dihydrate (CPPD) crystals in joint diseases plays an important role in the development of gouty arthritis and joint chondrocalcinosis, respectively (1, 2). Although many cell types are involved in the pathogenesis of these inflammatory joint diseases, polymorphonuclear neutrophils play a central role particularly during the acute phase of the arthritis. Indeed, the inflammatory episodes observed in natural gout or in acute crystal-induced synovitis have been associated with a major accumulation of neutrophils in both the synovium and the synovial fluid, and many of these cells encounter and charac- teristically phagocytose crystals (2, 3). In addition, crystal-in- duced experimental arthritis in dogs is markedly suppressed when neutrophils are depleted by antipolymorphonuclear leu- kocyte serum or cytotoxic drugs (4, 5). The activation of neutrophils by MSU and/or CPPD crys- tals leads to the production and secretion of several inflamma- tory mediators such as lysosomal enzymes (6, 7), oxygen-de- rived free radicals (8, 9), 5-lipoxygenase products ( 10), crystal- induced chemotactic factor ( 1 1, 12), and IL- 1 ( 13). These mediators may be responsible, at least in part, for the systemic manifestations associated with crystal-induced joint disorders. The signaling pathway(s) involved in the mediation of these responses to microcrystals have only recently begun to be in- vestigated. The addition of MSU and/or CPPD crystals to a suspension of human neutrophils leads to rapid increases in the cytoplasmic concentration of free calcium ( 10, 14), to the for- mation of inositol 1,4,5 triphosphate ( 14), to the activation of a phosphatidylcholine-specific phospholipase D (15), and to increases in the level of protein tyrosine phosphorylation ( 16). Two sets of results indicate that significant differences underlie the interaction of neutrophils with chemotactic factors and MSU or CPPD crystals. First, and somewhat indirectly, the responses to microcrystals have been found to be significantly more resistant to pertussis toxin than those of chemotactic fac- tors ( 10, 17, 18), thereby implying the utilization of different coupling systems. Second, the tyrosine phosphorylation pat- tern stimulated by microcrystals was observed to be character- istic of these agonists and to differ qualitatively as well as quan- titatively from that stimulated by a variety of soluble and partic- ulate stimuli ( 16). Among the therapeutic agents used in the treatment of acute arthritis, the usefulness of colchicine is relatively, al- though not exclusively, limited to crystal-associated rheumatic diseases such as gout ( 19). Although the effectiveness of colchi- cine has been reported as early as the sixth century (20), its precise mechanism of action is still obscure. Colchicine is thought to interfere with numerous neutrophil functions such 1. Abbreviations used in this paper: CPPD, calcium pyrophosphate dihydrate; LTB4, leukotriene B4; MSU, monosodium urate. 1722 Roberge, Gaudry, de Medicis, Lussier, Poubelle, and Naccache J. Clin. Invest. © The American Society for Clinical Investigation, Inc. 0021-9738/93/10/1722/08 $2.00 Volume 92, October 1993, 1722-1729