Tyrosine phosphorylation events play major roles in the initiation and regulation of several functional responses of human neutrophils stimulated by chemotactic factors such as the bacterially derived tripeptide formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe). However, the links between the G protein-coupled receptors, the activation of the tyrosine kinases, and the initiation of neutrophil functional responses remain unclear. In the present study we assessed the effects of a Btk inhibitor, leflunomide metabolite analog (LFM-A13), on neutrophils. LFM-A13 decreased the tyrosine phosphorylation induced by fMet-Leu-Phe and inhibited the production of superoxide anions and the stimulation of adhesion, chemotaxis, and phospholipase D activity. We observed a decreased accumulation of phosphatidylinositol-3,4,5-trisphosphate in response to fMet-Leu-Phe in LFM-A13-pretreated cells even though the inhibitor had no direct effect on the lipid kinase activity of the p110γ or p85/p110 phosphatidylinositol 3-kinases or on the activation of p110γ by fMet-Leu-Phe. The phosphorylation of Akt and of extracellular signal-regulated kinases 1/2 and p38 were similarly inhibited by LFM-A13. LFM-A13 also negatively affected the translocation of Rac-2, RhoA, ADP ribosylation factor-1, Tec, Bmx, and Btk induced by fMet-Leu-Phe. The results of this study provide evidence for an involvement of Btk and possibly other Tec kinase family members in the regulation of the functional responsiveness of human neutrophils and link these events, in part at least, to the modulation of levels of phosphatidylinositol-3,4,5-trisphosphate.

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The myeloid inhibitory receptor CLEC12A negatively regulates inflammation. Reduced CLEC12A expression enhances inflammation in CLEC12A knock-out mice with collagen antibody-induced arthritis. Moreover, CLEC12A internalisation augments human neutrophil activation. We thus postulated that CLEC12A expression on circulating myeloid cells of rheumatoid arthritis patients is associated with disease manifestations. Cell-surface, CLEC12A receptor expression was determined on circulating neutrophils and monocytes of eRA patients and of healthy donors. Generalized estimating equations model, Student’s t-test and Spearman’s correlations were performed to compare CLEC12A expression between groups and test its association with disease activity and clinical parameters. Plasma cytokines were measured by multiplex immunoassay. Patients with reduced neutrophil or monocyte CLEC12A expression at baseline and at 3 months have an increased simple disease activity index. Low baseline CLEC12A expression also correlates with a higher SDAI at 6 months. In contrast, positive correlations were observed between baseline CLEC12A expression and several cytokines. Moreover, neutrophil and monocyte CLEC12A expression is significantly higher in early rheumatoid arthritis patients at baseline than healthy controls. Circulating neutrophil and monocyte CLEC12A expression correlates with disease activity at baseline and is predictive of SDAI at later stages of the disease indicative of a regulatory role for CLEC12A in RA.

The inflammatory response in acute gouty arthritis is in large part a result of the interaction between neutrophils and monosodium urate (MSU) crystals. The tyrosine kinase Syk, which has been largely associated with the phagocytic response by Fc receptors and with spreading mediated by integrins, has been identified as one of the major proteins tyrosine-phosphorylated in human neutrophils upon stimulation by MSU crystals and is known to be mediated in part by the Fc receptor, CD16. This has led to the present examination of the implication of Syk in the activation pathways used by MSU crystals. The tyrosine-phosphorylation patterns induced by MSU crystals and by the ligation of CD16 were inhibited by piceatannol, which, conversely, only slightly delayed but did not diminish the peak of tyrosine phosphorylation induced by cross-linking CD32 or by the addition of fMet-Leu-Phe. Moreover, piceatannol inhibited the activity of Syk as monitored by in vitro kinase assays, by its in situ tyrosine phosphorylation, and by its activity toward exogenous substrates after stimulation by MSU crystals. We also measured the impact of piceatannol on the mobilization of calcium, the production of superoxide anions, and the activity of PLD stimulated by MSU crystals. We noted a distinct inhibition of all these responses by piceatannol. Finally, the morphological changes observed in neutrophils as characteristic of MSU crystal internalization were diminished significantly by piceatannol. The results obtained show that Syk plays a critical and central role in the signal-transduction pathways called upon by MSU crystals subsequent to their interaction with human neutrophils.