Selective blockade of NF-kappa B activity in airway immune cells inhibits the effector phase of experimental asthma

Knockout mice studies have revealed that NF-kappaB plays a critical role in Th2 cell differentiation and is therefore required for induction of allergic airway inflammation. However, the questions of whether NF-kappaB also plays a role in the effector phase of airway allergy and whether inhibiting NF-kappaB could have therapeutic value in the treatment of established asthma remain unanswered. To address these issues, we have assessed in OVA-sensitized wild-type mice the effects of selectively antagonizing NF-kappaB activity in the lungs during OVA challenge. Intratracheal administration of NF-kappaB decoy oligodeoxynucleotides to OVA-sensitized mice led to efficient nuclear transfection of airway immune cells, but not constitutive lung cells and draining lymph node cells, associated with abrogation of NF-kappaB activity in the airways upon OVA provocation. NF-kappaB inhibition was associated with strong attenuation of allergic lung inflammation, airway hyperresponsiveness, and local production of mucus, IL-5, IL-13, and eotaxin. IL-4 and OVA-specific IgE and IgG1 production was not reduced. This study demonstrates for the first time that activation of NF-kappaB in local immune cells is critically involved in the effector phase of allergic airway disease and that specific NF-kappaB inhibition in the lungs has therapeutic potential in the control of pulmonary allergy.     

Airway epithelium controls lung inflammation and injury through the NF-kappa B pathway

Although airway epithelial cells provide important barrier and host defense functions, a crucial role for these cells in development of acute lung inflammation and injury has not been elucidated. We investigated whether NF-kappaB pathway signaling in airway epithelium could decisively impact inflammatory phenotypes in the lungs by using a tetracycline-inducible system to achieve selective NF-kappaB activation or inhibition in vivo. In transgenic mice that express a constitutively active form of IkappaB kinase 2 under control of the epithelial-specific CC10 promoter, treatment with doxycycline induced NF-kappaB activation with consequent production of a variety of proinflammatory cytokines, high-protein pulmonary edema, and neutrophilic lung inflammation. Continued treatment with doxycycline caused progressive lung injury and hypoxemia with a high mortality rate. In contrast, inducible expression of a dominant inhibitor of NF-kappaB in airway epithelium prevented lung inflammation and injury resulting from expression of constitutively active form of IkappaB kinase 2 or Escherichia coli LPS delivered directly to the airways or systemically via an osmotic pump implanted in the peritoneal cavity. Our findings indicate that the NF-kappaB pathway in airway epithelial cells is critical for generation of lung inflammation and injury in response to local and systemic stimuli; therefore, targeting inflammatory pathways in airway epithelium could prove to be an effective therapeutic strategy for inflammatory lung diseases.     

The IkappaB kinase is a key factor in triggering influenza A virus-induced inflammatory cytokine production in airway epithelial cells

Influenza A viruses continue to represent a severe threat worldwide, causing large epidemics and pandemics responsible for thousands of deaths every year. Excessive inflammation due to overabundant production of proinflammatory cytokines by airway epithelial cells is considered an important factor in disease pathogenesis. Here we report that influenza A virus induced IkappaB kinase (IKK) activity in human airway epithelial A549 cells, resulting in persistent activation of nuclear factor-kappaB (NF-kappaB), a critical regulator of the inflammatory response. Although lung epithelial cells are highly sensitive to stimulation of the IKK/NF-kappaB pathway by influenza virus infection, NF-kappaB was not activated in several non-pulmonary cells permissive to the virus, indicating a cell-specific response. Moreover, NF-kappaB was not essential for virus replication but triggered the expression of proinflammatory cytokines in infected lung cells and was directly responsible for production of high levels of interleukin-8, a chemokine associated with influenza-induced inflammation and airway pathology. We also report that 9-deoxy-delta9,delta12-13,14-dihydro-prostaglandin D2, a cyclopentenone prostanoid with therapeutic efficacy against influenza in preclinical studies, was a powerful inhibitor of influenza virus-induced IKK activity and interleukin-8 production by human pulmonary cells. The results identify IKK as an important factor in triggering influenza virus-induced inflammatory reactions in pulmonary epithelium, suggesting novel therapeutic approaches in the treatment of influenza.     

NF-kappaB in breast cancer cells promotes osteolytic bone metastasis by inducing osteoclastogenesis via GM-CSF

Advanced breast cancers frequently metastasize to bone, resulting in osteolytic lesions, yet the underlying mechanisms are poorly understood. Here we report that nuclear factor-kappaB (NF-kappaB) plays a crucial role in the osteolytic bone metastasis of breast cancer by stimulating osteoclastogenesis. Using an in vivo bone metastasis model, we found that constitutive NF-kappaB activity in breast cancer cells is crucial for the bone resorption characteristic of osteolytic bone metastasis. We identified the gene encoding granulocyte macrophage-colony stimulating factor (GM-CSF) as a key target of NF-kappaB and found that it mediates osteolytic bone metastasis of breast cancer by stimulating osteoclast development. Moreover, we observed that the expression of GM-CSF correlated with NF-kappaB activation in bone-metastatic tumor tissues from individuals with breast cancer. These results uncover a new and specific role of NF-kappaB in osteolytic bone metastasis through GM-CSF induction, suggesting that NF-kappaB is a potential target for the treatment of breast cancer and the prevention of skeletal metastasis.     

CARMA1 is critical for the development of allergic airway inflammation in a murine model of asthma

CARMA1 has been shown to be important for Ag-stimulated activation of NF-kappaB in lymphocytes in vitro and thus could be a novel therapeutic target in inflammatory diseases such as asthma. In the present study, we demonstrate that mice with deletion in the CARMA1 gene (CARMA1(-/-)) do not develop inflammation in a murine model of asthma. Compared with wild-type controls, CARMA1(-/-) mice did not develop airway eosinophilia, had no significant T cell recruitment into the airways, and had no evidence for T cell activation in the lung or draining lymph nodes. In addition, the CARMA1(-/-) mice had significantly decreased levels of IL-4, IL-5, and IL-13, did not produce IgE, and did not develop airway hyperresponsiveness or mucus cell hypertrophy. However, adoptive transfer of wild-type Th2 cells into CARMA1(-/-) mice restored eosinophilic airway inflammation, cytokine production, airway hyperresponsiveness, and mucus production. This is the first demonstration of an in vivo role for CARMA1 in a disease process. Furthermore, the data clearly show that CARMA1 is essential for the development of allergic airway inflammation through its role in T lymphocytes, and may provide a novel means to inhibit NF-kappaB for therapy in asthma.     

Identification of an autoregulatory feedback pathway involving interleukin-1alpha in induction of constitutive NF-kappaB activation in pancreatic cancer cells

We previously reported that NF-kappaB is constitutively activated in most human pancreatic cancer tissues and cell lines but not in normal pancreatic tissues and immortalized pancreatic ductal epithelial cells. IkappaBalphaM-mediated inhibition of constitutive NF-kappaB activity in human pancreatic cancer cells suppressed tumorigenesis and liver metastasis in an orthotopic nude mouse model, suggesting that constitutive NF-kappaB activation plays an important role in pancreatic tumor progression and metastasis. However, the underlying mechanism by which NF-kappaB is activated in pancreatic cancer remains to be elucidated. In this study, we found that an autocrine mechanism accounts for the constitutive activation of NF-kappaB in metastatic human pancreatic cancer cell lines. Further investigation showed that interleukin-1alpha was the primary cytokine secreted by these cells that activates NF-kappaB. Neutralization of interleukin-1alpha activity suppressed the constitutive activation of NF-kappaB and the expression of its downstream target gene, urokinase-type plasminogen activator, in metastatic pancreatic cancer cell lines. Our results demonstrate that regulation of interleukin-1alpha expression is primarily dependent on AP-1 activity, which is in part induced by signaling pathways that are epidermal growth factor receptor-dependent and -independent. In conclusion, our findings suggest a possible mechanism for the constitutive activation of NF-kappaB in metastatic human pancreatic cancer cells and a possible missing mechanistic link between inflammation and cancer.     

Nicotine prevents the apoptosis induced by menadione in human lung cancer cells

Approximately 50% of long-term cigarette smokers die prematurely from the adverse effects of smoking, including on lung cancer and other illnesses. Nicotine is a main component in tobacco and has been implicated as a potential factor in the pathogenesis of human lung cancer. However, the mechanism of nicotine action in the development of lung cancer remains largely unknown. In the present study, we designed a nicotine-apoptosis system, by pre-treatment of nicotine making lung cancer cell A549 to be in a physiological nicotine environment, and observed that nicotine promoted cell proliferation and prevented the menadione-induced apoptosis, and exerts its role of anti-apoptosis by shift of apoptotic stage induced by menadione from late apoptotic stage to early apoptotic stage, in which NF-kappaB was up-regulated. Interference analysis of NF-kappaB in A549 cells showed that knock down of NF-kappaB resulted in apoptosis promotion and counteracted the protective effect of nicotine. The findings suggest that nicotine has potential effect in lung cancer genesis, especially in patients with undetectable early tumor development and development of specific NF-kappaB inhibitors would represent a potentially exciting new pharmacotherapy for tobacco-related lung cancer.     

Depletion of beta-arrestin-2 promotes tumor growth and angiogenesis in a murine model of lung cancer

Arrestins are adaptor/scaffold proteins that complex with activated and phosphorylated G protein-coupled receptor to terminate G protein activation and signal transduction. These complexes also mediate downstream signaling, independently of G protein activation. We have previously shown that beta-arrestin-2 (betaarr2) depletion promotes CXCR2-mediated cellular signaling, including angiogenesis and excisional wound closure. This study was designed to investigate the role of betaarr2 in tumorigenesis using a murine model of lung cancer. To that end, heterotopic murine Lewis lung cancer and tail vein metastasis tumor model systems in betaarr2-deficient mice (betaarr2(-/-)) and control littermates (betaarr2(+/+)) were used. betaarr2(-/-) mice exhibited a significant increase in Lewis lung cancer tumor growth and metastasis relative to betaarr2(+/+) mice. This correlated with decreased number of tumor-infiltrating lymphocytes but with elevated levels of the ELR(+) chemokines (CXCL1/keratinocyte-derived chemokine and CXCL2/MIP-2), vascular endothelial growth factor, and microvessel density. NF-kappaB activity was also enhanced in betaarr2(-/-) mice, whereas hypoxia-inducible factor-1alpha expression was decreased. Inhibition of CXCR2 or NF-kappaB reduced tumor growth in both betaarr2(-/-) and betaarr2(+/+) mice. NF-kappaB inhibition also decreased ELR(+) chemokines and vascular endothelial growth factor expression. Altogether, the data suggest that betaarr2 modulates tumorigenesis by regulating inflammation and angiogenesis through activation of CXCR2 and NF-kappaB.     

Curcumin regulates airway epithelial cell cytokine responses to the pollutant cadmium

Cadmium is a toxic metal present in the environment and its inhalation can lead to pulmonary disease such as lung cancer and chronic obstructive pulmonary disease. These lung diseases are characterized by chronic inflammation. Here we show that exposure of human airway epithelial cells to cadmium promotes a polarized apical secretion of IL-6 and IL-8, two pivotal pro-inflammatory cytokines known to play an important role in pulmonary inflammation. We also determined that two distinct pathways controlled secretion of these proinflammatory cytokines by human airway epithelial cells as cadmium-induced IL-6 secretion occurs via an NF-κB dependent pathway, whereas IL-8 secretion involves the Erk1/2 signaling pathway. Interestingly, the natural antioxidant curcumin could prevent both cadmium-induced IL-6 and IL-8 secretion by human airway epithelial cells. In conclusion, curcumin could be used to prevent airway inflammation due to cadmium inhalation.     

Stromal cell-derived factor-1 increase alphavbeta3 integrin expression and invasion in human chondrosarcoma cells

Chondrosarcoma is a type of highly malignant tumor with a potent capacity to invade locally and cause distant metastasis. Chondrosarcoma shows a predilection for metastasis to the lungs. The stromal cell-derived factor 1 (SDF-1), constitutively secreted by human lung epithelium cells, has been shown to function in a key role for recruitment of neutrophils. Here, we found that human chondrosarcoma tissues and chondrosarcoma cell lines had significant expression of CXCR4 (SDF-1 receptor), which was higher than normal cartilage and human chondrocyte. SDF-1alpha and lung epithelium cells conditioned medium (LECM) induced the invasiveness of chondrosarcoma cells. SDF-1 siRNA inhibited LECM-induced invasion of chondrosarcoma cells and SDF-1alpha also directly induced the cell surface expression of alphavbeta3 but not alpha2beta1 and alpha5beta1 integrin. Activations of ERK and NF-kappaB pathways after SDF-1 treatment was demonstrated, and SDF-1alpha-induced expression of alphavbeta3 integrin and invasion activity was inhibited by the specific inhibitor and mutant of ERK and NF-kappaB cascades. Taken together, our results indicate that lung derived-SDF-1alpha enhances the invasiveness of chondrosarcoma cells by increasing alphavbeta3 integrin expression through the CXCR4/ERK/NF-kappaB signal transduction pathway.     

IkappaB kinase is a critical regulator of chemokine expression and lung inflammation in respiratory syncytial virus infection

Respiratory syncytial virus (RSV) is the major etiologic agent of severe epidemic lower respiratory tract infections in infancy. Airway mucosal inflammation plays a critical role in the pathogenesis of RSV disease in both natural and experimental infections. RSV is among the most potent biological stimuli that induce the expression of inflammatory genes, including those encoding chemokines, but the mechanism(s) that controls virus-mediated airway inflammation in vivo has not been fully elucidated. Herein we show that the inoculation of BALB/c mice with RSV results in rapid activation of the multisubunit IkappaB kinase (IKK) in lung tissue. IKK transduces upstream activating signals into the rate-limiting phosphorylation (and proteolytic degradation) of IkappaBalpha, the inhibitory subunit that under normal conditions binds to the nuclear factor (NF)-kappaB complex and keeps it in an inactive cytoplasmic form. Mice treated intranasally with interleukin-10 or with a specific cell-permeable peptide that blocks the association of the catalytic subunit IKKbeta with the regulatory protein NEMO showed a striking reduction of lung NF-kappaB DNA binding activity, chemokine gene expression, and airway inflammation in response to RSV infection. These findings suggest that IKKbeta may be a potential target for the treatment of acute or chronic inflammatory diseases of the lung.     

Selective I kappa B kinase expression in airway epithelium generates neutrophilic lung inflammation

To determine whether NF-kappaB activation is sufficient to generate lung inflammation in vivo, we selectively expressed a constitutively active form of IkappaB kinase 1 (cIKK1) or IkappaB kinase 2 (cIKK2) in airway epithelium. After intratracheal administration of adenoviral vectors expressing cIKK1 or cIKK2 to transgenic reporter mice that express Photinus luciferase under the control of an NF-kappaB-dependent promoter, we detected significantly increased luciferase activity over time (up to 96 h). Compared with control mice treated with adenoviral vectors expressing beta-galactosidase, lung bioluminescence and tissue luciferase activity were increased in NF-kappaB reporter mice treated with adenovirus (Ad)-cIKK1 or Ad-cIKK2. NF-kappaB activation in lungs of Ad-cIKK1- and Ad-cIKK2-treated mice was confirmed by immunoblots for RelA and EMSA from lung nuclear protein extracts. Mice treated with Ad-cIKK1 or Ad-cIKK2 showed induction of mRNA expression of several chemokines and cytokines in lung tissue. In lung lavage fluid, mice treated with Ad-cIKK1 or Ad-cIKK2 showed elevated concentrations of NF-kappaB-dependent chemokines macrophage-inflammatory protein 2 and KC and increased numbers of neutrophils. Coadministration of adenoviral vectors expressing a transdominant inhibitor of NF-kappaB with Ad-cIKK1 or Ad-cIKK2 resulted in abrogated NF-kappaB activation and other parameters of lung inflammation, demonstrating that the observed inflammatory effects of Ad-cIKK1 and Ad-cIKK2 were dependent on NF-kappaB activation by these kinases. These data show that selective expression of IkappaB kinases in airway epithelium results in NF-kappaB activation, inflammatory mediator production, and neutrophilic lung inflammation. Therapies targeted to NF-kappaB in lung epithelium may be beneficial in treating inflammatory lung diseases.     

Peroxisome proliferator-activated receptor-gamma in cystic fibrosis lung epithelium

The pathophysiology of cystic fibrosis (CF) inflammatory lung disease is not well understood. CF airway epithelial cells respond to inflammatory stimuli with increased production of proinflammatory cytokines as a result of increased NF-kappaB activation. Peroxisome proliferator-activated receptor-gamma (PPARgamma) inhibits NF-kappaB activity and is reported to be reduced in CF. If PPARgamma participates in regulatory dysfunction in the CF lung, perhaps PPARgamma ligands might be useful therapeutically. Cell models of CF airway epithelium were used to evaluate PPARgamma expression and binding to NF-kappaB at basal and under conditions of inflammatory stimulation by Pseudomonas aeruginosa or TNFalpha/IL-1beta. An animal model of CF was used to evaluate the potential of PPARgamma agonists as therapeutic agents in vivo. In vitro, PPARgamma agonists reduced IL-8 and MMP-9 release from airway epithelial cells in response to PAO1 or TNFalpha/IL-1beta stimulation. Less NF-kappaB bound to PPARgamma in CF than normal cells, in two different assays; PPARgamma agonists abrogated this reduction. PPARgamma bound less to its target DNA sequence in CF cells. To test the importance of the reported PPARgamma inactivation by phosphorylation, we observed that inhibitors of ERK, but not JNK, were synergistic with PPARgamma agonists in reducing IL-8 secretion. In vivo, administration of PPARgamma agonists reduced airway inflammation in response to acute infection with P. aeruginosa in CF, but not wild-type, mice. In summary, PPARgamma inhibits the inflammatory response in CF, at least in part by interaction with NF-kappaB in airway epithelial cells. PPARgamma agonists may be therapeutic in CF.     

Enhanced airway reactivity and inflammation in A2A adenosine receptor-deficient allergic mice

A(2A) adenosine receptor (A(2A)AR) has potent anti-inflammatory properties, which may be important in the regulation of airway reactivity and inflammation. Inflammatory cells that possess A(2A)AR also produce nitrosative stress, which is associated with pathophysiology of asthma, so we hypothesized that A(2A)AR deficiency may lead to increased airway reactivity and inflammation through nitrosative stress. Thus the present study was carried out to investigate the role of A(2A)AR on airway reactivity, inflammation, NF-kappaB signaling, and nitrosative stress in A(2A)AR knockout (KO) and wild-type (WT) mice using our murine model of asthma. Animals were sensitized intraperitoneally on days 1 and 6 with 200 microg of ragweed, followed by aerosolized challenges with 0.5% ragweed on days 11, 12, and 13, twice a day. On day 14, airway reactivity to methacholine was assessed as enhanced pause (Penh) using whole body plethysmography followed by bronchoalveolar lavage (BAL) and lung collection for various analyses. Allergen challenge caused a significant decrease in expression of A(2A)AR in A(2A) WT sensitized mice, with A(2A)AR expression being undetected in A(2A) KO sensitized group leading to decreased lung cAMP levels in both groups. A(2A)AR deletion/downregulation led to an increase in Penh to methacholine and influx of total cells, eosinophils, lymphocytes, and neutrophils in BAL with highest values in A(2A) KO sensitized group. A(2A) KO sensitized group further had increased NF-kappaB expression and nitrosative stress compared with WT sensitized group. These data suggest that A(2A)AR deficiency leads to airway inflammation and airway hyperresponsiveness, possibly via involvement of nitrosative stress in this model of asthma.