Chemotactic Factor-Induced Recruitment and Activation of Tec Family Kinases in Human Neutrophils. II. Effects of LFM-A13, a Specific Btk Inhibitor

Abstract

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.

Interleukin-8-induced Priming of Neutrophil Oxidative Burst Requires Sequential Recruitment of NADPH Oxidase Components into Lipid Rafts

“…We have shown that IL-8 treatment induces membrane translocation of Btk. Furthermore, after adding LFM-A13, an inhibitor of Btk, we observed the inhibition of the IL-8-induced activation of the MAPK and Akt/PKB pathways reminiscent of previous results obtained after fMLP stimulation (31). LFM-A13 also inhibited both p47 phox and p67 phox phosphorylation induced by IL-8 treatment.…”

“…On the basis of the effects of the inhibitors tested (genistein and wortmannin), we showed that the IL-8-induced phosphorylation of both p47 phox and p67 phox depended on the upstream activation of tyrosine kinase and PI3K. Among the tyrosine kinases, the Tec kinases, and especially Btk has recently been shown to be involved in fMLP signaling, thus providing a direct link between G protein-coupled receptors and activation of the tyrosine kinases (31). Activation of the Tec kinases is mediated in part by the high affinity of their pleckstrin homology domains for phosphatidylinositol 3,4,5-P 3 (50), which is generated following the activation of PI3K.…”

“…4, A and B). Among the tyrosine kinases, the Tec kinases, and especially Btk, has been shown to contribute to fMLP-induced neutrophil responses by modulating the MAPK and PI3K/Akt/PKB pathways (31). The role of Btk during the IL-8 priming process was previously unknown.…”

Bruton tyrosine kinase is essential for botrocetin/VWF-induced signaling and GPIb-dependent thrombus formation in vivo

“…The role of the Tec family nonreceptor tyrosine kinases, Btk and Tec, in bt/VWF-induced signaling in platelets was investigated because of the close relationship between PI3K and Btk in signal transduction in B cells, 17,18 neutrophils, 39 and platelets 19,40 and because previous work from this laboratory demonstrated that bt/VWF-induced TxA2 production is inhibited by the PI3K selective inhibitor wortmannin. 14 Furthermore, Btk plays an important role in PLC␥ activation, [20][21][22][23] and PLC␥ is required for bt/VWFinduced TxA2 production.…”

Roles for phosphoinositide 3‐kinases, Bruton's tyrosine kinase, and Jun kinases in B lymphocyte chemotaxis and homing

“…We focused on three, WMN because of the known involvement of PI3K in lymphocyte chemotaxis [8,9], LFM-A13 because of the importance of Bruton's tyrosine kinase (Btk) in B lymphocyte function and recent demonstration of its role in neutrophil chemotaxis [22], and JNK because of its known role in the migration of other cell types [19,20].…”