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BACKGROUND Human rhinovirus (HRV) infections trigger exacerbations of asthma and chronic obstructive pulmonary disease. Nitric oxide (NO) inhibits HRV replication in human airway epithelial cells and suppresses HRV-induced epithelial production of several cytokines and chemokines. OBJECTIVE We sought to delineate the mechanisms by which NO inhibits HRV-induced epithelial production of CXCL10, a chemoattractant for type 1 T cells and natural killer cells. METHODS Primary human bronchial epithelial cells or cells of the BEAS-2B human bronchial epithelial cell line were exposed to HRV-16 in the presence or absence of the NO donor 3-(2-hydroxy-2-nitroso-1-propylhydrazino)-1-propanamine (PAPA NONOate). A cGMP analogue and an inhibitor of soluble guanylyl cyclase were used to examine the role of the cyclic guanosine monophosphate (cGMP) pathway in the actions of NO. BEAS-2B cells were transfected with CXCL10 promoter-luciferase constructs and the effects of PAPA NONOate were examined to study mechanisms of transcriptional regulation. Electrophoretic mobility shift assays were also used. RESULTS PAPA NONOate inhibited HRV-16-induced increases in CXCL10 mRNA and protein. Inhibition of CXCL10 production occurred through a cGMP-independent pathway. PAPA NONOate inhibited HRV-16-induced CXCL10 transcription by blocking nuclear translocation, binding, or both of both nuclear factor kappaB and IFN response factors (IRFs) to their respective recognition elements in the CXCL10 promoter. CONCLUSIONS NO inhibits HRV-16-induced production of CXCL10 by inhibiting viral activation of nuclear factor kappaB and of IRFs, including IRF-1, through a cGMP-independent pathway. The broad-ranging inhibition of HRV-induced epithelial cytokine and chemokine production by NO suggests a potential therapeutic utility of NO donors in viral exacerbations of asthma and chronic obstructive pulmonary disease.

Rationale: Airway remodeling, a characteristic feature of asthma, begins in early life. Recurrent human rhinovirus (HRV) infections are a potential inciting stimulus for remodeling. One component of airway remodeling is an increase in airway smooth muscle cell mass with a greater proximity of the airway smooth muscle cells (ASMC) to the airway epithelium. We asked whether human bronchial epithelial cells infected with HRV produced mediators that are chemotactic for ASMC. Methods: ASMC migration was investigated using the modified Boyden Chamber and the xCELLigence Real-Time Cell Analyzer. Multiplex bead analysis was used to measure HRV-induced epithelial chemokine release. The chemotactic effects of CCL5, CXCL8 and CXCL10 were also examined. Results: Supernatants from HRV-infected epithelial cells caused ASMC chemotaxis. Pre-treatment of ASMC with pertussis toxin abrogated chemotaxis, as did treatment with formoterol, forskolin, or 8-bromo-cAMP. CCL5, CXCL8, and CXCL10 were most up-regulated chemokines produced by HRV-infected airway epithelial cells. When recombinant CCL5, CXCL8 and CXCL10 were used at levels found in epithelial supernatants they induced ASMC chemotaxis similar to that seen with epithelial cell supernatants. When examined individually, CCL5 was the most effective chemokine in causing ASMC migration and treatment of supernatant from HRV-infected epithelial cells with anti-CCL5 antibodies significantly attenuated ASMC migration. Conclusion: These findings suggest that HRV-induced CCL5 can induce ASMC chemotaxis, and thus may contribute to the pathogenesis of airway remodeling in asthmatic patients.

Human rhinovirus (HRV) infections can trigger exacerbations of lower airway diseases. Infection of airway epithelial cells induces production of a number of proinflammatory chemokines that may exacerbate airway inflammation, including CXCL10, a chemoattractant for type 1 lymphocytes and NK cells. Primary human bronchial epithelial cells and the BEAS-2B human bronchial epithelial cell line were used to examine the role of MAPK pathways in HRV-16-induced production of CXCL10. Surprisingly, PD98059 and U0126, two inhibitors of the MEK1/2-ERK MAPK pathway, significantly enhanced HRV-16-induced CXCL10 mRNA and protein. This enhancement was not seen with IFN-beta-induced production of CXCL10. Studies using small interfering RNA revealed that knockdown of MEK1, but not MEK2, was associated with enhanced HRV-induced CXCL10 production. Promoter construct studies revealed that PD98059 and U0126 enhanced HRV-16-induced transcriptional activation of CXCL10. HRV-16-induced promoter activation was regulated by two NF-kappaB binding sites, kappaB1 and kappaB2, and by an IFN-stimulated response element. Inhibitors of the MEK1/2-ERK pathway did not alter HRV-16-induced activation of tandem repeat kappaB1 or kappaB2 constructs, nor did they alter HRV-16-induced nuclear translocation/binding of NF-kappaB to either kappaB1 or kappaB2 recognition sequences. Furthermore, PD98059 and U0126 did not alter phosphorylation or degradation of IkappaBalpha. In contrast, inhibitors of the MEK1/2-ERK pathway, and small interfering RNA knockdown of MEK1, enhanced nuclear translocation/binding of IFN regulatory factor (IRF)-1 to the IFN-stimulated response element recognition sequence in HRV-16 infected cells. We conclude that activation of MEK1 selectively down-regulates HRV-16-induced expression of CXCL10 via modulation of IRF-1 interactions with the gene promoter in human airway epithelial cells.

Human rhinovirus (HRV) infections up-regulate proinflammatory mediators and growth factors that are associated with exacerbations of inflammatory airway diseases, such as asthma and chronic obstructive pulmonary disease (COPD). Matrix metalloproteinase (MMP)-9 was shown to be increased in the airways of patients with asthma and COPD. We sought to determine whether HRV infection modulated the expression of MMP-9 and its highest-affinity inhibitor, the tissue inhibitor of metalloproteinase (TIMP)-1, and we explored the mechanism by which this modulation occurs. In vitro studies, using RT-PCR, ELISA, zymography, and a fluorescent activity assay, demonstrated that MMP-9 mRNA, protein, and activity were increased upon infection with HRV, whereas TIMP-1 mRNA and protein remained unchanged. These results were verified in vivo, using nasal lavage samples obtained from subjects with confirmed rhinovirus infections. Human rhinovirus infections were shown to up-regulate NF-kappaB, and NF-kappaB has also been reported to play a role in the expression of MMP-9. We therefore investigated the role of NF-kappaB in HRV-induced MMP-9 expression. Using two inhibitors of IkappaBalpha kinase beta, we observed a concentration-dependent decrease in HRV-induced MMP-9 expression. The role of NF-kappaB in HRV-induced MMP-9 expression was further confirmed using MMP-9 promoter luciferase constructs, which demonstrated that an NF-kappaB site at -620/-607 base pairs was necessary for HRV-induced MMP-9 expression. Electrophoretic mobility shift assays and supershift assays confirmed the nuclear translocation and binding of p50/p65 NF-kappaB subunits to an MMP-9-specific NF-kappaB oligonucleotide. This increase in MMP-9 may be a mechanism by which rhinovirus infections contribute to airway inflammation and, potentially, to airway remodeling.