Cytokine (tumor necrosis factor, IL-6, and IL-8) production by respiratory syncytial virus-infected human alveolar macrophages.

Human alveolar macrophages (AM) are susceptible to infection with respiratory syncytial virus (RSV), but the infection is abortive after the initial cycles of virus replication. We have investigated if RSV infection of AM results in the production of cytokines TNF, IL-6, and IL-8, all of which may modulate inflammatory and immune responses to the virus, as well as may directly protect respiratory epithelial cells against spread of infection. Within 1 h after interaction with RSV, increased mRNA levels were found for all three cytokines. Peak expression of the mRNAs occurred at 3 to 6 h. The virus most effectively induced TNF mRNA expression greater than IL-6 mRNA greater than IL-8 mRNA, as compared to cytokine mRNA expression induced by bacterial endotoxin. Inactivated virus was almost as effective as live virus in inducing and maintaining increased IL-6 and IL-8 mRNA over 16 h, whereas live infectious RSV was necessary for maintaining TNF mRNA expression over the same time. Protein concentrations of the different cytokines in the supernatants of infected AM reflected the increased levels of mRNA in the cells. Despite the high levels of cytokines with possible antiviral activity (TNF and IL-6) in the AM supernatants, neither supernatants nor rTNF when added to bronchial epithelial cells protected them from infection with RSV. However, TNF, IL-1, and RSV, but not IL-6, induced IL-8 and IL-6 mRNA expression by the bronchial epithelial cells suggesting that cytokines produced by RSV-infected AM may be more important in modulating the inflammatory response in infection than directly interfering with virus infection/replication of airway epithelium.     

IFN-gamma amplifies IL-6 and IL-8 responses by airway epithelial-like cells via indoleamine 2,3-dioxygenase

Respiratory viral infections increase inflammatory responses to concurrent or secondary bacterial challenges, thereby worsening disease outcome. This potentiation of inflammation is explained at least in part by IFN-gamma promoting increased sensitivity to TNF-alpha and LPS. We sought to determine whether and, if so, how IFN-gamma can modulate proinflammatory responses to TNF-alpha and LPS by epithelial cells, which are key effector cells in the airways. Preincubation of airway epithelial-like NCI-H292 cells with IFN-gamma resulted in a hyperresponsive IL-6 and IL-8 production to TNF-alpha and LPS. The underlying mechanism involved the induction of indoleamine 2,3-dioxygenase, which catabolized the essential amino acid, tryptophan. Depletion of tryptophan led to stabilization of IL-6 and IL-8 mRNA and increased IL-6 and IL-8 responses, whereas supplementing tryptophan largely restored these changes. This novel mechanism may be implicated in enhanced inflammatory responses to bacterial challenges following viral infection.     

MAPK signaling pathways regulate IL-8 mRNA stability and IL-8 protein expression in cystic fibrosis lung epithelial cell lines

Cystic fibrosis (CF) is characterized by a massive proinflammatory phenotype in the lung, caused by mutations in the CFTR gene. IL-8 and other proinflammatory mediators are elevated in the CF airway, and the immediate mechanism may depend on disease-specific stabilization of IL-8 mRNA in CF lung epithelial cells. MAPK signaling pathways impact directly on IL-8 protein expression in CF cells, and we have hypothesized that the mechanism may also involve stabilization of the IL-8 mRNA. To test this hypothesis, we have examined the effects of pharmacological and molecular inhibitors of p38, and downstream MK2, ERK1/2, and JNK, on stability of IL-8 mRNA in CF lung epithelial cells. We previously showed that tristetraprolin (TTP) was constitutively low in CF and that raising TTP destabilized the IL-8 mRNA. We therefore also tested these effects on CF lung epithelial cells stably expressing TTP. TTP binds to AU-rich elements in the 3'-UTR of the IL-8 mRNA. We find that inhibition of p38 and ERK1/2 reduces the stability of IL-8 mRNA in parental CF cells. However, neither intervention further lowers TTP-dependent destabilization of IL-8 mRNA. By contrast, inhibition of the JNK-2 pathway has no effect on IL-8 mRNA stability in parental CF cell, but rather increases the stability of the message in cells expressing high levels of TTP. However, we find that inhibition of ERK1/2 or p38 leads to suppression of the effect of JNK-2 inhibition on IL-8 mRNA stability. These data thus lend support to our hypothesis that constitutive MAPK signaling and proteasomal activity might also contribute, along with aberrantly lower TTP, to the proinflammatory phenotype in CF lung epithelial cells by increasing IL-8 mRNA stability and IL-8 protein expression.     

IL-6 protein production by airway epithelial(-like) cells disabled in IL-6 mRNA degradation

IL-6 mRNA and protein expression in human airway epithelial-like H292 cells depends on rapid, but regulable IL-6 mRNA degradation. We restricted IL-6 mRNA degradation by partially inhibiting protein synthesis and studied the IL-6 response. Despite partial inhibition of protein synthesis, IL-6 protein production was increased and prolonged. Furthermore, the threshold concentration for stimuli of IL-6 protein production decreased and the dose-response curves became steeper. Similar findings were obtained with primary human bronchial epithelial cells. This exaggerated production may apply to other proteins encoded by labile mRNA and is likely to occur during viral infection of airway epithelial cells.     

Haemophilus influenzae stimulates ICAM-1 expression on respiratory epithelial cells

Epithelial cells interact directly with bacteria in the environment and play a critical role in airway defense against microbial pathogens. In this study, we examined the response of respiratory epithelial cells to infection with nontypable Haemophilus influenzae. Using an in vitro cell culture model, we found that epithelial cell monolayers released significant quantities of IL-8 and expressed increased levels of ICAM-1 mRNA and surface protein in response to H. influenzae. In contrast, levels of IL-1beta, TNF-alpha, and MHC class I were not significantly affected, suggesting preferential activation of a specific subset of epithelial genes directed toward defense against bacteria. Induction of ICAM-1 required direct bacterial interaction with the epithelial cell surface and was not reproduced by purified H. influenzae lipooligosaccharide. Consistent with a functional role for this response, induction of ICAM-1 by H. influenzae mediated increased neutrophil adherence to the epithelial cell surface. Furthermore, in an in vivo murine model of airway infection with H. influenzae, increased epithelial cell ICAM-1 expression coincided with increased chemokine levels and neutrophil recruitment in the airway. These results indicate that ICAM-1 expression on human respiratory epithelial cells is induced by epithelial cell interaction with H. influenzae and suggest that an ICAM-1-dependent mechanism can mediate neutrophil adherence to these cells independent of inflammatory mediator release by other cell types. Direct induction of specific epithelial cell genes (such as ICAM-1 and IL-8) by bacterial infection may allow for rapid and efficient innate defense in the airway.     

Regulation of interleukin-8 via an airway epithelial signaling cascade

Airways function as an innate immune organ against airborne bacteria that are inhaled and deposited in airways. One of the mechanisms of host defense is to recruit neutrophils into airways to clear the invaders. Airway epithelial cells produce neutrophil chemoattractant interleukin (IL)-8 in response to invading bacteria. In this study we show a signaling pathway on the plasma surface of human airway epithelial NCI-H292 cells that regulate IL-8 production in response to a model inflammatory stimulus, phorbol 12-myristate 13-acetate, and a pathophysiological stimulus, gram-negative bacterial lipopolysaccharide. First, we show that EGF receptor (EGFR) and MAP kinase ERK1/2 are involved in IL-8 expression by these stimuli. Second, we show that EGFR ligand transforming growth factor (TGF)-alpha mediates IL-8 production. Third, we show that tumor necrosis factor-alpha-converting enzyme (TACE) is required for IL-8 production by cleaving EGFR proligand proTGF-alpha into soluble TGF-alpha, activating EGFR. Last, we show that dual oxidase 1 (Duox1), a homolog of NADPH oxidase in airways, mediates TACE activation and IL-8 expression via generation of reactive oxygen species. In summary, we describe a signaling pathway, Duox1-TACE-TGF-alpha-EGFR, on the surface of airway epithelial (NCI-H292) cells that mediates airway epithelial defense against bacterial infection by producing IL-8. This pathway, which also regulates mucin production in human airways, provides mechanisms for killing foreign organisms and for their clearance.     

IL-15 up-regulates iNOS expression and NO production by gingival epithelial cells

To investigate the biological activity of epithelial cells in view of host defense, we analyzed the mRNA expression of inducible NOS (iNOS) as well as NO production by human gingival epithelial cells (HGEC) stimulated with IL-15. RT-PCR analysis revealed that HGEC expressed IL-15 receptor alpha-chain mRNA. In addition, stimulation with IL-15 enhanced iNOS expression by HGEC through an increase of both mRNA and protein levels. Moreover, IL-15 up-regulated the production of NO(2)(-)/NO(3)(-), a NO-derived stable end product, from HGEC. The enhanced NO production by IL-15 was inhibited by AMT, an iNOS-specific inhibitor. These results suggest that IL-15 is a potent regulator of iNOS expression by HGEC and involved in innate immunity in the mucosal epithelium.     

IL-33 mediates inflammatory responses in human lung tissue cells

IL-33 is a member of the IL-1 family and mediates its biological effects via the ST2 receptor, which is selectively expressed on Th2 cells and mast cells. Although polymorphic variation in ST2 is strongly associated with asthma, it is currently unclear whether IL-33 acts directly on lung tissue cells at sites of airway remodeling. Therefore, we aimed to identify the IL-33-responsive cells among primary human lung tissue cells. ST2 mRNA was expressed in both endothelial and epithelial cells but not in fibroblasts or smooth muscle cells. Correspondingly, IL-33 promoted IL-8 production by both endothelial and epithelial cells but not by fibroblasts or smooth muscle cells. Transfection of ST2 small interference RNA into both endothelial and epithelial cells significantly reduced the IL-33-dependent upregulation of IL-8, suggesting that IL-33-mediated responses in these cells occur via the ST2 receptor. Importantly, Th2 cytokines, such as IL-4, further enhanced ST2 expression and function in both endothelial and epithelial cells. The IL-33-mediated production of IL-8 by epithelial cells was almost completely suppressed by corticosteroid treatment. In contrast, the effect of corticosteroid treatment on the IL-33-mediated responses of endothelial cells was only partial. IL-33 induced activation of both ERK and p38 MAPK in endothelial cells but only ERK in epithelial cells. p38 MAPK was required for the IL-33-mediated responses of endothelial cells, whereas ERK was required for IL-33-mediated IL-8 production by epithelial cells. Taken together, these findings suggest that IL-33-mediated inflammatory responses of lung tissue cells may be involved in the chronic allergic inflammation of the asthmatic airway.     

IL-4 induces mucin gene expression and goblet cell metaplasia in vitro and in vivo

Goblet cell metaplasia and mucus hypersecretion are important features in the pathogenesis of asthma. The cytokine IL-4 has been shown to play a role in animal models of asthma, where it induces Th2 lymphocyte differentiation and B lymphocyte IgE class switch. IL-4 has also been implicated in the differentiation of goblet cells via effects on lymphocytes and eosinophils. In this study we hypothesized that IL-4 induces airway epithelial cell mucin gene expression and mucous glycoconjugate production by direct action on these cells. In vitro, cultured airway epithelial cells (NCI-H292) expressed IL-4R constitutively, and IL-4 (10 ng/ml) induced MUC2 gene expression and mucous glycoconjugate production. In vivo, mouse airway epithelial cells expressed IL-4R constitutively, and IL-4 (250 ng) increased MUC5 gene expression and Alcian blue/periodic acid-Schiff-positive staining at 24 h; IL-4 did not increase inflammatory cell numbers in airway tissue or in bronchoalveolar lavage. TNF-alpha and IL-1beta levels in bronchoalveolar lavage were not increased in response to IL-4 instillation. These results indicate that airway epithelial cells express IL-4R constitutively and that IL-4 directly induces the differentiation of epithelium into mucous glycoconjugate-containing goblet cells.     

8-Oxoguanine-DNA glycosylase 1 deficiency modifies allergic airway inflammation by regulating STAT6 and IL-4 in cells and in mice

8-Oxoguanine-DNA glycosylase (OGG-1) is a base excision DNA repair enzyme; however, its function in modulating allergic diseases remains undefined. Using OGG-1 knockout (KO) mice, we show that this protein affects allergic airway inflammation after sensitization and challenge by ovalbumin(OVA). OGG-1 KO mice exhibited less inflammatory cell infiltration and reduced oxidative stress in the lungs after OVA challenge compared to WT mice. The KO phenotype included decreased IL-4, IL-6, IL-10, and IL-17 in lung tissues. In addition, OGG-1 KO mice showed decreased expression and phosphorylation of STAT6 as well as NF-κB. Down-regulation of OGG-1 by siRNA lowered ROS and IL-4 levels but increased IFN-γ production in cultured epithelial cells after exposure to house dust mite extracts. OGG-1 may affect the levels of oxidative stress and proinflammatory cytokines during asthmatic conditions. OGG-1 deficiency negatively regulates allergen-induced airway inflammatory response.     

Differential regulation of hyaluronan-induced IL-8 and IP-10 in airway epithelial cells

Airway epithelium is emerging as a regulator of local inflammation and immune responses. However, the cellular and molecular mechanisms responsible for the immune modulation by these cells have yet to be fully elucidated. At the cellular level, the hallmarks of airway inflammation are mucus gland hypertrophy with excess mucus production, accumulation of inflammatory mediators, inflammation in the airway walls and lumen, and breakdown and turnover of the extracellular matrix. We demonstrate that fragments of the extracellular matrix component hyaluronan induce inflammatory chemokine production in primary airway epithelial cells grown at an air-liquid interface. Furthermore, hyaluronan fragments use two distinct molecular pathways to induce IL-8 and IFN-gamma-inducible protein 10 (IP-10) chemokine expression in airway epithelial cells. Hyaluronan-induced IL-8 requires the MAP kinase pathway, whereas hyaluronan-induced IP-10 utilizes the NF-kappaB pathway. The induction is specific to low-molecular-weight hyaluronan fragments as other glycosaminoglycans do not induce IL-8 and IP-10 in airway epithelial cells. We hypothesize that not only is the extracellular matrix a target of destruction in airway inflammation but it plays a critical role in perpetuating inflammation through the induction of cytokines, chemokines, and modulatory enzymes in epithelial cells. Furthermore, hyaluronan, by inducing IL-8 and IP-10 by distinct pathways, provides a unique target for differential regulation of key inflammatory chemokines.