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°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 SykK396R 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.

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.

Spleen tyrosine kinase (Syk) is an immunoregulatory tyrosine kinase that was identified originally in leukocytes. It is a key regulator of innate immunity as well as hematopoietic cell differentiation and proliferation. A role for Syk in regulating normal cellular functions in nonhematopoietic cells is increasingly recognized. We have shown previously robust Syk expression in airway epithelium, where it regulates the early inflammatory response to human rhinovirus (HRV) infections, and HRV cell entry by clathrinmediated endocytosis. To test the hypothesis that Syk plays a role in modulating airway epithelial cell proliferation, migration, and production of vascular endothelial growth factor and interleukin-8, we studied the BEAS-2B human bronchial epithelial cell line and primary human airway epithelia from normal and asthmatic donors using Syk-specific pharmacologic inhibitors and small interfering RNA. Using an in vitro "wounding" model, we demonstrated significant impairment of "wound" closure after treatment with the Syk inhibitors N4-(2,2-dimethyl-3-oxo-4H-pyrid[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediamine (R406) and 2-[7-(3,4-dimethoxyphenyl)-imidazo[1,2-c]pyrimidin-5-ylamino]-nicotinamide dihydrochloride (BAY61-3606), overexpression of the kinase-inactive Syk K396R mutant, and Syk knockdown by small interfering RNA. HRV infection also impaired wound healing, an effect that was partly Syk-dependent because wound healing was impaired further when HRV infection occurred in the presence of Syk inhibition. Further investigation of potential regulatory mechanisms revealed that inhibition of Syk suppressed HRV-induced vascular endothelial growth factor expression while promoting the activation of caspase-3, a mediator of epithelial cell apoptosis. Together, these results indicate that Syk plays a role in promoting epithelial cell proliferation and migration, while mitigating the effects of apoptosis.

Human rhinovirus (HRV), cause of the common cold, is a leading cause of exacerbations of asthma and chronic obstruction pulmonary disease (COPD). Binding of HRV to ICAM (intercellular adhesion molecule)-1, its major receptor, induces a profound inflammatory response from airway epithelial cells. My laboratory has identified Syk tyrosine kinase to be an early regulator of HRV-ICAM-1 signalling: Syk mediates replication-independent p38 mitogen-activated protein (MAP) kinase and phosphatidyl-inositol 3 (PI3)-kinase activation, interleukin (IL)-8 expression, as well as HRV internalization via clathrin-mediated endocytosis. Syk activation is accompanied by formation of a protein complex consisting of ICAM-1, ezrin and Syk at the plasma membrane. However, the molecular mechanisms that regulate this process are not understood. In this report, we investigated the role of the Syk-SH2 domains and the ezrin ITAM (immuno-tyrosine activation motif)-like motif in HRV-induced cell activation using the human BEAS-2B airway epithelial cells. Our observations suggest that the ezrin-ITAM plays a role in Syk recruitment and activation by binding to the Syk tandem SH2 domains, as originally described in the canonical ITAM-mediating signal transduction pathway in hematopoietic cells. This report is the first to demonstrate ITAM-mediated signaling in non-hematopoietic cells, suggesting that this signaling paradigm may be more ubiquitous than previously recognized.

Spleen Tyrosine Kinase (Syk) is widely expressed in the immune system and functions in the transmission of inflammatory signals via ITAM-bearing cell surface receptors. The broad expression pattern and importance of Syk in regulating innate immunity and the inflammatory response have led to significant interest from the pharmaceutical industry to developing anti-Syk therapeutics for the treatment of inflammatory disorders such as allergic rhinitis and rheumatoid arthritis. While the function and regulation of Syk has been well-described in leukocytes, where its primary role is an early transducer of signaling following immunoreceptor engagement, Syk has recently been described in non-immune cells, such as the airway epithelium, that also play an important role in mediating the inflammatory response. This manuscript will focus on the expression and function of Syk in the context of inflammatory lung diseases, and review recent data that have demonstrated novel roles for Syk in airway epithelial cells, particularly its role in mediating the human rhinovirus (HRV) induced inflammatory response and viral cell entry. In addition, data describing the efficacy of novel Syk inhibitors in the management of inflammatory diseases in animal models and early clinical trials are also reviewed.