Neutrophils 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.

Kovács et al. examine the role of the Src family kinases Hck, Fgr, and Lyn in immune cell–mediated inflammation. Using arthritis and skin inflammation models, the authors show that mice lacking hematopoietic Hck, Fgr, and Lyn are protected from these inflammatory diseases, showing loss of myeloid cell recruitment and lack of inflammatory mediator production. Unexpectedly, the three kinases are dispensable for the intrinsic migratory ability of myeloid cells. These finding may have clinical implications in rheumatic and skin diseases.

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Neutrophils are terminally differentiated cells with limited transcriptional activity. The biological function of their gene expression changes is poorly understood. CARD9 regulates transcription during antifungal immunity but its role in sterile inflammation is unclear. Here we show that neutrophil CARD9 mediates pro-inflammatory chemokine/cytokine but not lipid mediator release during non-infectious inflammation. Genetic deficiency of CARD9 suppresses autoantibody-induced arthritis and dermatitis in mice. Neutrophil-specific deletion of CARD9 is sufficient to induce that phenotype. Card9−/− neutrophils show defective immune complex-induced gene expression changes and pro-inflammatory chemokine/cytokine release but normal LTB4 production and other short-term responses. In vivo deletion of CARD9 reduces tissue levels of pro-inflammatory chemokines and cytokines but not LTB4. The CARD9-mediated signalling pathway involves Src-family kinases, Syk, PLCγ2, Bcl10/Malt1 and NFκB. Collectively, CARD9-mediated gene expression changes within neutrophils play important roles during non-infectious inflammation in vivo and CARD9 acts as a divergence point between chemokine/cytokine and lipid mediator release.

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.

IntroductionDasatinib (BMS-354825) is a second-generation oral tyrosine kinase inhibitor primarily used as a second-line treatment in imatinib-resistant chronic myeloid leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia. 1 Similar to imatinib, dasatinib also inhibits the Abl kinase and the Bcr-Abl fusion protein, although with a different molecular mechanism of action. 2,3 In addition to Abl and Bcr-Abl, dasatinib also inhibits several additional kinases, including Src and Btk family members, c-Kit, PDGFR, and Eph receptors. 3,4 In addition to its effect on malignant cells, dasatinib also inhibits certain functions of normal cells of various hematopoietic lineages, including T lymphocytes, 5 natural killer cells, 6 basophils, 7 platelets, 8,9 and osteoclasts. 10 However, no information is available on the effect of dasatinib on neutrophils, the most abundant circulating leukocytes.Neutrophils are short-lived, terminally differentiated phagocytic cells that provide the first line of defense against bacterial and fungal pathogens, but also contribute to the development of various acute and chronic inflammatory diseases. 11,12 Neutrophil activation occurs through several cell-surface receptors (integrins, Fc receptors, G-protein-coupled receptors, and cytokine and innate immune receptors) that activate complex intracellular signal transduction events leading to cellular responses such as adhesion, migration, respiratory burst, granule release, phagocytosis, and bacterial killing.Several genetic and pharmacologic studies have indicated that protein tyrosine kinases play critical roles in neutrophil activation by various cell-surface receptors. 13 Src-family kinases are involved in neutrophil functions triggered through integrins [14][15][16] or formylpeptide receptors. 15,17 Pharmacologic studies have suggested a role for Abl in integrin-mediated activation, 18 L-selectin shedding, 19 and respiratory burst 20 of neutrophils. We and others have identified critical roles for Syk in various neutrophil-activation pathways. 16,[21][22][23][24] The lack of information on the effect of dasatinib on neutrophils, the putative role of Src-family kinases and c-Abl in neutrophil activation, and the inhibition of certain neutrophil functions by dasatinib in a kinase inhibitor screening study (K.F., T.V., G. Kéri, and A.M., unpublished observations, December 2010) prompted us to perform a detailed analysis of the effect of dasatinib on human neutrophil functions. Our results indicate that dasatinib exerts a robust inhibitory effect on various inflammationrelated functions of mature human neutrophils. Methods Neutrophil isolation and inhibitor treatmentHuman neutrophils were isolated from venous blood of healthy volunteers by Ficoll or Percoll gradient centrifugation, followed by hypotonic lysis of RBCs. 17,25 Cells were resuspended in Ca 2ϩ -and Mg 2ϩ -free HBSS supplemented with 20mM HEPES, pH 7.4, and kept at room temperature until use.Dasatinib (Ͼ 99% pure) was obtained from Selleck Chemicals and its pu...

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 Ca2+-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.

β2-integrins of neutrophils play a critical role in innate immune defense but they also participate in tissue destruction during autoimmune inflammation. p190RhoGAP, a regulator of Rho-family small GTPases, is required for integrin signal transduction in fibroblasts. Prior studies have also suggested a role for p190RhoGAP in β2 integrin signaling in neutrophils. To directly test that possibility, we have generated a novel targeted mutation completely disrupting the p190RhoGAP-encoding gene in mice. p190RhoGAP deficiency led to perinatal lethality and defective neural development, precluding the analysis of neutrophil functions in adult p190RhoGAP−/− animals. This was overcome by transplantation of fetal liver cells from p190RhoGAP−/− fetuses into lethally irradiated wild type recipients. Neutrophils from such p190RhoGAP−/− bone marrow chimeras developed normally and expressed normal levels of various cell surface receptors. Though p190RhoGAP−/− neutrophils showed moderate reduction of β2 integrin-mediated adherent activation, they showed mostly normal migration in β2-integrin-dependent in vitro and in vivo assays and normal β2 integrin-mediated killing of serum-opsonized S. aureus and E. coli. A neutrophil- and β2 integrin-dependent transgenic model of the effector phase of autoimmune arthritis also proceeded normally in p190RhoGAP−/− bone marrow chimeras. In contrast, all the above responses were completely blocked in CD18−/− neutrophils or CD18−/− bone marrow chimeras. These results suggest that p190RhoGAP likely does not play a major indispensable role in β2 integrin-mediated in vitro and in vivo neutrophil functions or the effector phase of experimental autoimmune arthritis.