Analysis of intracellular tyrosine phosphorylation in circulating neutrophils as a rapid assay for the in vivo effect of oral tyrosine kinase inhibitors

Tyrosine kinases are crucial signaling components of diverse biological processes and are major therapeutic targets in various malignancies and immune-mediated disorders. A critical step of development of novel tyrosine kinase inhibitors is the transition from the confirmation of the in vitro effects of drug candidates to the analysis of their in vivo efficacy. To facilitate this transition, we have developed a rapid in vivo assay for the analysis of the effect of oral tyrosine kinase inhibitors on basal tyrosine phosphorylation of circulating mouse neutrophils. The assay uses a single drop of peripheral blood without sacrificing the mice. Flow cytometry using intracellular staining by fluorescently labeled anti-phosphotyrosine antibodies revealed robust basal tyrosine phosphorylation in resting circulating neutrophils. This signal was abrogated by the use of isotype control antibodies or by pre-saturation of the anti-phosphotyrosine antibodies with soluble phosphotyrosine amino acids or tyrosine-phosphorylated peptides. Basal tyrosine phosphorylation was dramatically reduced in neutrophils of triple knockout mice lacking the Src-family tyrosine kinases Hck, Fgr, and Lyn. Neutrophil tyrosine phosphorylation was also abrogated by oral administration of the Abl/Src-family inhibitor dasatinib, a clinically used anti-leukemic agent. Detailed dose-response and kinetic studies revealed half-maximal reduction of neutrophil tyrosine phosphorylation by 2.9 mg/kg dasatinib, with maximal reduction observed 2 h after inhibitor administration. Taken together, our assay allows highly efficient analysis of the in vivo effect of orally administered tyrosine kinase inhibitors, and may be used as a suitable alternative to other existing approaches

Importance of Fc Receptor γ-Chain ITAM Tyrosines in Neutrophil Activation and in vivo Autoimmune Arthritis

Activating Fcγ receptors associated with Fc receptor γ-chain (FcRγ) are critical for mediating neutrophil effector functions in immune complex-mediated autoimmune diseases. FcRγ contains ITAM tyrosines and the in vivo role of these tyrosines has not been defined in neutrophils and arthritis. In this study, the in vivo functions of FcRγ ITAM tyrosines were characterized using wild type and ITAM tyrosine mutant (Y65F/Y76F) transgenic mice crossed to an FcRγ-deficient genetic background. FcRγ-deficient neutrophils showed undetectable cell surface expression of the activating Fcγ receptor IV, defective immune complex-induced superoxide production, degranulation and spreading. Although the re-expression of both the wild type and the ITAM tyrosine mutant (Y65F/Y76F) FcRγ could restore activating Fcγ receptor expression of FcRγ-deficient neutrophils, only the wild type transgenic form could mediate Fcγ receptor-dependent effector functions. In contrast, neutrophils carrying ITAM tyrosine mutant FcRγ were unable to produce superoxide, mediate degranulation and perform active spreading. In addition, our results confirmed the protection of FcRγ-deficient mice from autoimmune arthritis. Importantly, the presence of the wild type FcRγ transgene, in contrast to the ITAM tyrosine mutant transgene, partially reversed autoimmune arthritis development. The reversing effect of the wild type transgene was even more robust when animals carried the wild type transgene in a homozygous form. Collectively, FcRγ ITAM tyrosines play a critical role in the induction of neutrophil effector responses, the initiation and progression of an autoantibody-induced experimental arthritis in vivo, indicating a signaling, rather than just a receptor stabilizing function of the molecule

MASP-1 Induces a Unique Cytokine Pattern in Endothelial Cells: A Novel Link between Complement System and Neutrophil Granulocytes

Microbial infection urges prompt intervention by the immune system. The complement cascade and neutrophil granulocytes are the predominant contributors to this immediate anti-microbial action. We have previously shown that mannan-binding lectin-associated serine protease-1 (MASP-1), the most abundant enzyme of the complement lectin pathway, can induce p38-MAPK activation, NFkappaB signaling, and Ca(2+)-mobilization in endothelial cells. Since neutrophil chemotaxis and transmigration depends on endothelial cell activation, we aimed to explore whether recombinant MASP-1 (rMASP-1) is able to induce cytokine production and subsequent neutrophil chemotaxis in human umbilical vein endothelial cells (HUVEC). We found that HUVECs activated by rMASP-1 secreted IL-6 and IL-8, but not IL-1alpha, IL-1ra, TNFalpha and MCP-1. rMASP-1 induced dose-dependent IL-6 and IL-8 production with different kinetics. rMASP-1 triggered IL-6 and IL-8 production was regulated predominantly by the p38-MAPK pathway. Moreover, the supernatant of rMASP-1-stimulated HUVECs activated the chemotaxis of neutrophil granulocytes as an integrated effect of cytokine production. Our results implicate that besides initializing the complement lectin pathway, MASP-1 may activate neutrophils indirectly, via the endothelial cells, which link these effective antimicrobial host defense mechanisms

Targeting vascular endothelial growth factor receptor 2 and protein kinase d1 related pathways by a multiple kinase inhibitor in angiogenesis and inflammation related processes in vitro.

Emerging evidence suggests that the vascular endothelial growth factor receptor 2 (VEGFR2) and protein kinase D1 (PKD1) signaling axis plays a critical role in normal and pathological angiogenesis and inflammation related processes. Despite all efforts, the currently available therapeutic interventions are limited. Prior studies have also proved that a multiple target inhibitor can be more efficient compared to a single target one. Therefore, development of novel inflammatory pathway-specific inhibitors would be of great value. To test this possibility, we screened our molecular library using recombinant kinase assays and identified the previously described compound VCC251801 with strong inhibitory effect on both VEGFR2 and PKD1. We further analyzed the effect of VCC251801 in the endothelium-derived EA.hy926 cell line and in different inflammatory cell types. In EA.hy926 cells, VCC251801 potently inhibited the intracellular activation and signaling of VEGFR2 and PKD1 which inhibition eventually resulted in diminished cell proliferation. In this model, our compound was also an efficient inhibitor of in vitro angiogenesis by interfering with endothelial cell migration and tube formation processes. Our results from functional assays in inflammatory cellular models such as neutrophils and mast cells suggested an anti-inflammatory effect of VCC251801. The neutrophil study showed that VCC251801 specifically blocked the immobilized immune-complex and the adhesion dependent TNF-alpha -fibrinogen stimulated neutrophil activation. Furthermore, similar results were found in mast cell degranulation assay where VCC251801 caused significant reduction of mast cell response. In summary, we described a novel function of a multiple kinase inhibitor which strongly inhibits the VEGFR2-PKD1 signaling and might be a novel inhibitor of pathological inflammatory pathways

Neutrophil cell surface receptors and their intracellular signal transduction pathways

AbstractNeutrophils play a critical role in the host defense against bacterial and fungal infections, but their inappropriate activation also contributes to tissue damage during autoimmune and inflammatory diseases. Neutrophils express a large number of cell surface receptors for the recognition of pathogen invasion and the inflammatory environment. Those include G-protein-coupled chemokine and chemoattractant receptors, Fc-receptors, adhesion receptors such as selectins/selectin ligands and integrins, various cytokine receptors, as well as innate immune receptors such as Toll-like receptors and C-type lectins. The various cell surface receptors trigger very diverse signal transduction pathways including activation of heterotrimeric and monomeric G-proteins, receptor-induced and store-operated Ca2+ signals, protein and lipid kinases, adapter proteins and cytoskeletal rearrangement. Here we provide an overview of the receptors involved in neutrophil activation and the intracellular signal transduction processes they trigger. This knowledge is crucial for understanding how neutrophils participate in antimicrobial host defense and inflammatory tissue damage and may also point to possible future targets of the pharmacological therapy of neutrophil-mediated autoimmune or inflammatory diseases

Tyrosine kinase signaling pathways in neutrophils

Neutrophils play a critical role in antimicrobial host defense, but their improper activation also contributes to inflammation-induced tissue damage. Therefore, understanding neutrophil biology is important for the understanding, diagnosis, and therapy of both infectious and inflammatory diseases. Neutrophils express a large number of cell-surface receptors that sense extracellular cues and trigger various functional responses through complex intracellular signaling pathways. During the last several years, we and others have shown that tyrosine kinases play a critical role in those processes. In particular, Src-family and Syk tyrosine kinases couple Fc-receptors and adhesion receptors (integrins and selectins) to various neutrophil effector functions. This pathway shows surprising similarity to lymphocyte antigen receptor signaling and involves various other enzymes (e.g. PLCγ2), exchange factors (e.g. Vav-family members) and adapter proteins (such as ITAM-containing adapters, SLP-76, and CARD9). Those mediators trigger various antimicrobial functions and play a critical role in coordinating the inflammatory response through the release of inflammatory mediators, such as chemokines and LTB4. Interestingly, however, tyrosine kinases have a limited direct role in the migration of neutrophils to the site of inflammation. Here, we review the role of tyrosine kinase signaling pathways in neutrophils and how those pathways contribute to neutrophil activation in health and disease