Syk is downstream of intercellular adhesion molecule-1 and mediates human rhinovirus activation of p38 MAPK in airway epithelial cells

The airway epithelium is the primary target of inhaled pathogens such as human rhinovirus (HRV). Airway epithelial cells express ICAM-1, the major receptor for HRV. HRV binding to ICAM-1 mediates not only viral entry and replication but also a signaling cascade that leads to enhanced inflammatory mediator production. The specific signaling molecules and pathways activated by HRV-ICAM-1 interactions are not well characterized, although studies in human airway epithelia implicate a role for the p38 MAPK in HRV-induced cytokine production. In the current study, we report that Syk, an important immunoregulatory protein tyrosine kinase, is highly expressed by primary and cultured human airway epithelial cells and is activated in response to infection with HRV16. Biochemical studies revealed that ICAM-1 engagement by HRV and cross-linking Abs enhanced the coassociation of Syk with ICAM-1 and ezrin, a cytoskeletal linker protein. In polarized airway epithelial cells, Syk is diffusely distributed in the cytosol under basal conditions but, following engagement of ICAM-1 by cross-linking Abs, is recruited to the plasma membrane. The enhanced Syk-ICAM-1 association following HRV exposure is accompanied by Syk phosphorylation. ICAM-1 engagement by HRV and cross-linking Abs also induced phosphorylation of p38 in a Syk-dependent manner, and conversely, knockdown of Syk by short interfering (si)RNA substantially diminished p38 activation and IL-8 gene expression. Taken together, these observations identify Syk as an important mediator of the airway epithelial cell inflammatory response by modulating p38 phosphorylation and IL-8 gene expression following ICAM-1 engagement by HRV.     

Involvement of Syk kinase in TNF-induced nitric oxide production by airway epithelial cells

We have recently found that Syk is widely expressed in lung epithelial cells (EC) and participates in beta1 integrin signaling. In this study, we assessed the role of Syk in regulation of NO production. Stimulation of human bronchial EC line HS-24 by TNF caused an increased expression of inducible nitric oxide synthase (iNOS). Inhibition of Syk using siRNA or piceatannol down-regulated the iNOS expression and reduced NO production. This effect occurred in EC simultaneously stimulated via beta1 integrins, suggesting that TNF and beta1 integrins provide co-stimulatory signals. Inhibition of Syk down-regulated TNF-induced p38 and p44/42 MAPK phosphorylation and nuclear translocation of p65 NF-kappaB. Thus, TNF-induced activation of pro-inflammatory signaling in EC leading to enhanced expression of iNOS and NO production was dependent on Syk. Syk-mediated signaling regulates NO production at least partly via activating the MAPK cascade. Understanding the role of Syk in airway EC may help in developing new therapeutic tools for inflammatory lung disorders.     

Syk associates with clathrin and mediates phosphatidylinositol 3-kinase activation during human rhinovirus internalization

Human rhinovirus (HRV) causes the common cold. The most common acute infection in humans, HRV is a leading cause of exacerbations of asthma and chronic obstruction pulmonary disease because of its ability to exacerbate airway inflammation by altering epithelial cell biology upon binding to its receptor, ICAM-1. ICAM-1 regulates not only viral entry and replication but also signaling pathways that lead to inflammatory mediator production. We recently demonstrated the Syk tyrosine kinase to be an important mediator of HRV-ICAM-1 signaling: Syk regulates replication-independent p38 MAPK activation and IL-8 expression. In leukocytes, Syk regulates receptor-mediated internalization via PI3K. Although PI3K has been shown to regulate HRV-induced IL-8 expression and clathrin-mediated endocytosis of HRV, the role of airway epithelial Syk in this signaling pathway is not known. We postulated that Syk regulates PI3K activation and HRV endocytosis in the airway epithelium. Using confocal microscopy and immunoprecipitation, we demonstrated recruitment of the normally cytosolic Syk to the plasma membrane upon HRV16-ICAM-1 binding, along with Syk-clathrin coassociation. Subsequent incubation at 37 degrees C to permit internalization revealed redistribution of Syk to punctate structures resembling endosomes and colocalization with HRV16. Internalized HRV was not detected in cells overexpressing the kinase inactive Syk(K396R) mutant, indicating that kinase activity was necessary for endocytosis. HRV-induced PI3K activation was dependent on Syk; Syk knockdown by small interfering RNA significantly decreased phosphorylation of the PI3K substrate Akt. Together, these data reveal Syk to be an important mediator of HRV endocytosis and HRV-induced PI3K activation.     

Beta2-integrin, LFA-1, and TCR/CD3 synergistically induce tyrosine phosphorylation of focal adhesion kinase (pp125(FAK)) in PHA-activated T cells

Complete T cell activation requires not only a first signal via TCR/CD3 engagement but also a costimulatory signal through accessory receptors such as CD2, CD28, or integrins. Focal adhesion kinase, pp125(FAK) (FAK), was previously shown to be localized in focal adhesions in fibroblasts and to be involved in integrin-mediated cellular activation. Although signaling through beta1- or beta3-integrins induces tyrosine phosphorylation of FAK, there has been no evidence that activation of T cells through the beta2-integrin, LFA-1, involves FAK. We report here that crosslinking of LFA-1 induces tyrosine phosphorylation of FAK in PHA-activated T cells. Moreover, cocrosslinking with anti-LFA-1 mAb and suboptimal concentration of anti-CD3 mAb markedly increases tyrosine phosphorylation of FAK in an antibody-concentration-dependent and time-kinetics-dependent manner compared with stimulation through CD3 alone, which correlates well with enhanced proliferation of PHA-activated T cells. Furthermore, LFA-1beta costimulation with CD3 induces tyrosine phosphorylation of Syk associated with FAK. These results indicate, for the first time, that signals mediated by LFA-1 can regulate FAK, suggesting that LFA-1-mediated T cell costimulation may be involved in T cell activation at least partially through FAK.     

Src-dependent Syk activation controls CD69-mediated signaling and function on human NK cells

CD69 C-type lectin receptor represents a functional triggering molecule on activated NK cells, capable of directing their natural killing function. The receptor-proximal signaling pathways activated by CD69 cross-linking and involved in CD69-mediated cytotoxic activity are still poorly understood. Here we show that CD69 engagement leads to the rapid and selective activation of the tyrosine kinase Syk, but not of the closely related member of the same family, ZAP70, in IL-2-activated human NK cells. Our results indicate the requirement for Src family kinases in the CD69-triggered activation of Syk and suggest a role for Lck in this event. We also demonstrate that Syk and Src family tyrosine kinases control the CD69-triggered tyrosine phosphorylation and activation of phospholipase Cgamma2 and the Rho family-specific exchange factor Vav1 and are responsible for CD69-triggered cytotoxicity of activated NK cells. The same CD69-activated signaling pathways are also observed in an RBL transfectant clone, constitutively expressing the receptor. These data demonstrate for the first time that the CD69 receptor functionally couples to the activation of Src family tyrosine kinases, which, by inducing Syk activation, initiate downstream signaling pathways and regulate CD69-triggered functions on human NK cells.     

Proinflammatory and Th2-derived cytokines modulate CD40-mediated expression of inflammatory mediators in airway epithelia: implications for the role of epithelial CD40 in airway inflammation

Cytokines produced by activated macrophages and Th2 cells within the lung play a key role in asthma-associated airway inflammation. Additionally, recent studies suggest that the molecule CD40 modulates lung immune responses. Because airway epithelial cells can act as immune effector cells through the expression of inflammatory mediators, the epithelium is now considered important in the generation of asthma-associated inflammation. Therefore, the goal of the present study was to examine the effects of proinflammatory and Th2-derived cytokines on the function of CD40 in airway epithelia. The results show that airway epithelial cells express CD40 and that engagement of epithelial CD40 induces a significant increase in expression of the chemokines RANTES, monocyte chemoattractant protein (MCP-1), and IL-8 and the adhesion molecule ICAM-1. Cross-linking epithelial CD40 had no effect on expression of the adhesion molecule VCAM-1. The proinflammatory cytokines TNF-alpha and IL-1beta and the Th2-derived cytokines IL-4 and IL-13 modulated the positive effects of CD40 engagement on inflammatory mediator expression in airway epithelial cells. Importantly, CD40 ligation enhanced the sensitivity of airway epithelial cells to the effects of TNF-alpha and/or IL-1beta on expression of RANTES, MCP-1, IL-8, and VCAM-1. In contrast, neither IL-4 nor IL-13 modified the effects of CD40 engagement on the expression of RANTES, MCP-1, IL-8, or VCAM-1; however, both IL-4 and IL-13 attenuated the effects of CD40 cross-linking on ICAM-1 expression. Together, these findings suggest that interactions between CD40-responsive airway epithelial cells and CD40 ligand+ leukocytes, such as activated T cells, eosinophils, and mast cells, modulate asthma-associated airway inflammation.     

Blockade of focal clustering and active conformation in beta 2-integrin-mediated adhesion of eosinophils to intercellular adhesion molecule-1 caused by transduction of HIV TAT-dominant negative Ras

We transduced dominant negative (dn) HIV TAT-Ras protein into mature human eosinophils to determine the signaling pathways and mechanism involved in integrin-mediated adhesion caused by cytokine, chemokine, and chemoattractant stimulation. Transduction of TAT-dnRas into nondividing eosinophils inhibited endogenous Ras activation and extracellular signal-regulated kinase (ERK) phosphorylation caused by IL-5, eotaxin-1, and fMLP. IL-5, eotaxin-1, or fMLP caused 1) change of Mac-1 to its active conformation and 2) focal clustering of Mac-1 on the eosinophil surface. TAT-dnRas or PD98059, a pharmacological mitogen-activated protein/ERK kinase inhibitor, blocked both focal surface clustering of Mac-1 and the change to active conformational structure of this integrin assessed by the mAb CBRM1/5, which binds the activation epitope. Eosinophil adhesion to the endothelial ligand ICAM-1 was correspondingly blocked by TAT-dnRas and PD98059. As a further control, we used PMA, which activates ERK phosphorylation by postmembrane receptor induction of protein kinase C, a mechanism which bypasses Ras. Neither TAT-dnRas nor PD98059 blocked eosinophil adhesion to ICAM-1, up-regulation of CBRM1/5, or focal surface clustering of Mac-1 caused by PMA. In contrast to beta(2)-integrin adhesion, neither TAT-dnRas nor PD98059 blocked the eosinophil adhesion to VCAM-1. Thus, a substantially different signaling mechanism was identified for beta(1)-integrin adhesion. We conclude that H-Ras-mediated activation of ERK is critical for beta(2)-integrin adhesion and that Ras-protein functions as the common regulator for cytokine-, chemokine-, and G-protein-coupled receptors in human eosinophils.     

Activation of macrophage CD8: pharmacological studies of TNF and IL-1 beta production

Previously, we demonstrated that rat macrophages express CD8 and that Ab to CD8 stimulates NO production. We confirm that CD8 is expressed by rat macrophages and extend understanding of its functional significance. Activation of CD8 alpha (OX8 Ab) on alveolar macrophages stimulated mRNA expression for TNF and IL-1 beta and promoted TNF and IL-1 beta secretion. Similarly, OX8 Ab (CD8 alpha) stimulated NR8383 cells to secrete TNF, IL-1 beta, and NO. Activation of CD8 beta (Ab 341) on alveolar macrophages increased mRNA expression for TNF and IL-1 beta and stimulated secretion of TNF, but not IL-1 beta. Interestingly, anti-CD8 Abs did not stimulate IFN-gamma or PGE2 production, or phagocytosis by macrophages. OX8 (CD8 alpha)-induced TNF and IL-1 beta production by macrophages was blocked by inhibitors of protein tyrosine kinase(s), PP1, and genistein, but not by phosphatidylinositol-3 kinase inhibitor, wortmannin. Moreover, OX8 stimulated protein tyrosine kinase activity in NR8383 cells. Further analysis of kinase dependence using antisense to Syk kinase demonstrated that TNF, but not IL-1 beta, stimulation by CD8 alpha is Syk dependent. By contrast, protein kinase C inhibitor Ro 31-8220 had no effect on OX8-induced TNF production, whereas OX8-induced IL-1 beta production was blocked by Ro 31-8220. Thus, there are distinct signaling mechanisms involved in CD8 alpha (OX8)-induced TNF and IL-1 beta production. In summary, macrophages express CD8 molecules that, when activated, stimulate TNF and IL-1 beta expression, probably through mechanisms that include activation of Src and Syk kinases and protein kinase C. These findings identify a previously unknown pathway of macrophage activation likely to be involved in host defense and inflammation.     

Aerosolized Syk antisense suppresses Syk expression, mediator release from macrophages, and pulmonary inflammation

Syk protein tyrosine kinase (PTK) is involved in signaling in leukocytes. In macrophages, Fcgamma-receptor cross-linking induces Syk PTK phosphorylation and activation, resulting in Syk-dependent events required for phagocytosis and mediator release. We hypothesized that Syk antisense oligodeoxynucleotides (ASO) delivered by aerosol to rat lungs in vivo would depress Syk PTK expression, mediator release from alveolar macrophages, and Syk-dependent pulmonary inflammation. RT-PCR and RT-in situ PCR demonstrated that aerosolized Syk ASO administration reduced Syk mRNA expression from alveolar macrophages compared with cells isolated from sham-treated rats. Western blot analysis confirmed that Syk PTK expression was reduced after Syk ASO treatment. Compared with sham-treated rats (scrambled oligodeoxynucleotide), Syk ASO treatment suppressed Fcgamma-receptor-mediated nitric oxide (86.0 +/- 8.3%) and TNF (73.1 +/- 3.1%) production by alveolar macrophages stimulated with IgG-anti-IgG complexes. In contrast, Fcgamma-receptor-induced IL-1beta release was unaffected by Syk ASO treatment. Additionally, Syk ASO suppressed Ag-induced pulmonary inflammation, suggesting that Syk ASO may prove useful as an anti-inflammatory therapy in disorders such as asthma.     

Epidermal growth factor receptor signaling to Erk1/2 and STATs control the intensity of the epithelial inflammatory responses to rhinovirus infection

Rhinovirus infection is the most common cause of acute exacerbations of inflammatory lung diseases, such as asthma and chronic obstructive pulmonary disease, where it provokes steroid refractory and abnormally intense neutrophilic inflammation that can be life threatening. Epidermal growth factor receptor (EGFR) expression correlates with disease severity and neutrophil infiltration in these conditions. However, the role of EGFR signaling in rhinovirus infection is unknown. We measured the key determinants of neutrophilic inflammation interleukin (IL)-8 and ICAM-1 in rhinovirus (RV16 serotype)-infected bronchial epithelial cells, BEAS-2B. RV16 infection stimulated IL-8 and ICAM-1 expression, which was further elevated (2-fold) by transient up-regulation of EGFR levels. Detection of viral RNA by quantitative real time PCR confirmed that enhanced expression was not associated with increased viral replication. EGFR ligands (epiregulin, amphiregulin, and heparin-binding epidermal growth factor) were induced by RV16 infection, and inhibition of metalloproteases responsible for ligand shedding partially suppressed this response. The EGFR inhibitor AG1478, completely blocked IL-8 and ICAM-1 expression to basal levels, as did the specific Erk1/2 inhibitor U0126. The p38 mitogen-activated protein kinase inhibitor SB203580 blocked IL-8 secretion but not ICAM-1 expression, whereas the PI3K inhibitor wortmannin was ineffective in both responses. Kinase inactive K721R EGFR, which is selectively deficient in STAT signaling, reversed RV16 responses associated with EGFR overexpression. In conclusion, RV16 infection rapidly promotes induction of EGFR ligands and utilizes EGFR signaling to increase IL-8 and ICAM-1 levels. These results suggest that targeting EGFR may provide a selective therapy that dampens neutrophil-driven inflammation without compromising essential antiviral pathways mediated by pathogen recognition receptors such as TLR3.     

Notch, epidermal growth factor receptor, and beta1-integrin pathways are coordinated in neural stem cells

Notch1 and beta1-integrins are cell surface receptors involved in the recognition of the niche that surrounds stem cells through cell-cell and cell-extracellular matrix interactions, respectively. Notch1 is also involved in the control of cell fate choices in the developing central nervous system (Lewis, J. (1998) Semin. Cell Dev. Biol. 9, 583-589). Here we report that Notch and beta1-integrins are co-expressed and that these proteins cooperate with the epidermal growth factor receptor in neural progenitors. We describe data that suggests that beta1-integrins may affect Notch signaling through 1) physical interaction (sequestration) of the Notch intracellular domain fragment by the cytoplasmic tail of the beta1-integrin and 2) affecting trafficking of the Notch intracellular domain via caveolin-mediated mechanisms. Our findings suggest that caveolin 1-containing lipid rafts play a role in the coordination and coupling of beta1-integrin, Notch1, and tyrosine kinase receptor signaling pathways. We speculate that this will require the presence of the adequate beta1-activating extracellular matrix or growth factors in restricted regions of the central nervous system and namely in neurogenic niches.     

Outer membrane protein A from Klebsiella pneumoniae activates bronchial epithelial cells: implication in neutrophil recruitment

Aside from its mechanical barrier function, bronchial epithelium plays an important role both in the host defense and in the pathogenesis of inflammatory airway disorders. To investigate its role in lung defense, the effect of a bacterial cell wall protein, the outer membrane protein A from Klebsiella pneumoniae (kpOmpA) on bronchial epithelial cells (BEC) was evaluated on adhesion molecule expression and cytokine production. Moreover, the potential implication of this mechanism in kpOmpA-induced lung inflammation was also determined. Our in vitro studies demonstrated that kpOmpA strongly bound to BEAS-2B cells, a human BEC line, and to BEC primary cultures, resulting in NF-kappaB signaling pathway activation. Exposure to kpOmpA increased ICAM-1 mRNA and cell surface expression, as well as the secretion of IL-6, CXC chemokine ligand (CXCL)1, CXCL8, C-C chemokine ligand 2, CXCL10 by BEAS-2B cells, and BEC primary cultures (p < 0.005). We analyzed in vivo the consequences of intratracheal injection of kpOmpA to BALB/c mice. In kpOmpA-treated mice, a transient neutrophilia (with a maximum at 24 h) was observed in bronchoalveolar lavage and lung sections. In vivo kpOmpA priming induced bronchial epithelium activation as evaluated by ICAM-1 and CXCL1 expression, associated with the secretion of CXCL1 and CXCL5 in bronchoalveolar lavage fluids. In the lung, an increased level of the IL-6, CXCL1, CXCL5, CXCL10 mRNA was observed with a maximum at 6 h. These data showed that kpOmpA is involved in host defense mechanism by its ability to activate not only APC but also BEC, resulting in a lung neutrophilia.     

A critical role for Syk protein tyrosine kinase in Fc receptor-mediated antigen presentation and induction of dendritic cell maturation

Dendritic cells (DCs) are the only APCs capable of initiating adaptive immune responses. The initiation of immune responses requires that DCs 1) internalize and present Ags; and 2) undergo a differentiation process, called "maturation", which transforms DCs into efficient APCs. DC maturation may be initiated by the engagement of different surface receptors, including certain cytokine receptors (such as TNFR), Toll-like receptors, CD40, and FcRs. The early activation events that link receptor engagement and DC maturation are not well characterized. We found that FcR engagement by immune complexes induced the phosphorylation of Syk, a protein tyrosine kinase acting immediately downstream of FcRs. Syk was dispensable for DC differentiation in vitro and in vivo, but was strictly required for immune complexes internalization and subsequent Ag presentation to T lymphocytes. Importantly, Syk was also required for the induction of DC maturation and IL-12 production after FcR engagement, but not after engagement of other surface receptors, such as TNFR or Toll-like receptors. Therefore, protein tyrosine phosphorylation by Syk represents a novel pathway for the induction of DC maturation.     

Inhibition of allergic inflammation in the airways using aerosolized antisense to Syk kinase

Activation of the protein tyrosine kinase Syk is an early event that follows cross-linking of Fc gamma R and Fc epsilon R, leading to the release of biologically active molecules in inflammation. We reported previously that aerosolized Syk antisense oligodeoxynucleotides (ASO) depresses Syk expression in inflammatory cells, the release of mediators from alveolar macrophages, and pulmonary inflammation. To study the effect of Syk ASO in allergic inflammation and airway hyperresponsiveness, we used the Brown Norway rat model of OVA-induced allergic asthma. Syk ASO, delivered in a liposome, carrier/lipid complex by aerosol to rats, significantly inhibited the Ag-induced inflammatory cell infiltrate in the bronchoalveolar space, decreasing both neutrophilia and eosinophilia. The number of eosinophils in the lung parenchyma was also diminished. Syk ASO also depressed up-regulation of the expression of beta(2) integrins, alpha(4) integrin, and ICAM-1 in bronchoalveolar lavage leukocytes and reversed the Ag-induced decrease in CD62L expression on neutrophils. Furthermore, the increase in TNF levels in bronchoalveolar lavage following Ag challenge was significantly inhibited. Syk ASO also suppressed Ag-mediated contraction of the trachea in a complementary model. Thus, aerosolized Syk ASO suppresses many of the central components of allergic asthma and inflammation and may provide a new therapeutic approach.     

The mechanism of IL-5 signal transduction

Cytokines are important regulators ofhematopoiesis. They exert their actions by binding to specificreceptors on the cell surface. Interleukin-5 (IL-5) is a criticalcytokine that regulates the growth, activation, and survival ofeosinophils. Because eosinophils play a seminal role in thepathogenesis of asthma and allergic diseases, an understanding of thesignal transduction mechanism of IL-5 is of paramount importance. TheIL-5 receptor is a heterodimer of - and -subunits. The-subunit is specific, whereas the -subunit is common to IL-3,IL-5, and granulocyte/macrophage colony-stimulating factor (GM-CSF)receptors and is crucial for signal transduction. It has been shownthat there are two major signaling pathways of IL-5 in eosinophils.IL-5 activates Lyn, Syk, and JAK2 and propagates signals through theRas-MAPK and JAK-STAT pathways. Studies suggest that Lyn, Syk, and JAK2tyrosine kinases and SHP-2 tyrosine phosphatase are important foreosinophil survival. In contrast to their survival-promoting activity,Lyn and JAK2 appear to have no role in eosinophil degranulation orexpression of surface adhesion molecules. Raf-1 kinase, on the otherhand, is critical for eosinophil degranulation and adhesion moleculeexpression. Btk is involved in IL-5 stimulation of B cell function.However, it does not appear to be important for eosinophil function.Thus a clear segregation of signaling molecules based on theirfunctional importance is emerging. This review describes the signaltransduction mechanism of the IL-3/GM-CSF/IL-5 receptor system andcompares and contrasts IL-5 signaling between eosinophils and B cells.

     

Tyrosine kinase Syk associates with toll-like receptor 4 and regulates signaling in human monocytic cells

Toll-like receptor 4 (TLR4) induces an innate immune response in mammals by recognizing lipopolysaccharide (LPS), a component of the cell wall of Gram-negative bacteria. In this study, we show that tyrosine kinase Syk constitutively associates with TLR4 in THP-1 cells. As previously reported in peripheral blood mononuclear cells, TLR4 gets inducibly tyrosine phosphorylated upon LPS engagement in THP-1 cells. Piceatannol, a pharmacological inhibitor of the tyrosine kinase Syk, abrogates TLR4 tyrosine phosphorylation at low doses. The kinetics of TLR4 tyrosine phosphorylation in THP-1 cells coincides with an early wave of Syk tyrosine phosphorylation. Additionally, serine threonine kinase interleukin-1 (IL1) receptor-associated kinase 1 (IRAK-1) is transiently recruited to the complex containing adaptor molecule MyD88, TLR4 and Syk within 1 min of LPS engagement and dissociates by 30 min. Finally, the inhibition of Syk with piceatannol has no effect on LPS-mediated release of cytokines IL6, IL1beta, tumor necrosis factor-alpha, neither on chemokines macrophage inhibitory protein (MIP)1alpha, MIP1beta, monocyte chemoattractant protein -1, IL8, Groalpha and RANTES. However, IL10 and IL12p40 releases are significantly inhibited. Our findings implicate Syk as a novel modulator of LPS-mediated TLR4 responses in human monocytic cells and shed insight into the kinetics of early complex formation upon LPS engagement.