Distinct mechanism of Helicobacter pylori-mediated NF-kappa B activation between gastric cancer cells and monocytic cells

NF-kappaB is a critical regulator of genes involved in inflammation. Gastric epithelial cells and macrophages are considered the main sources of pro-inflammatory cytokines. We investigated NF-kappaB activation by Helicobacter pylori in MKN45 gastric epithelial cells and THP-1 monocytic cells. Although, cag pathogenicity island (PAI)-positive H. pylori (wild type) activated NF-kappaB in both cells, isogenic mutant of cagE (DeltacagE) activated it only in THP-1 cells. Supernatant from the wild type culture could activate NF-kappaB in THP-1 cells but not in MKN45 cells. High density cDNA array analysis revealed that mRNA expression of NF-kappaB-regulated genes such as interleukin (IL)-8, tumor necrosis factor-alpha (TNFalpha), and IL-1beta was significantly up-regulated by the wild type in both cells, whereas it was up-regulated by DeltacagE only in THP-1 cells. Experiments using CD14-neutralizing antibody and IL-1 receptor-associated kinase (IRAK) assay showed that both wild type and DeltacagE H. pylori activated NF-kappaB through CD14 and IRAK in THP-1 cells but not in MKN45 cells. Macrophages from C3H/HeJ mice carrying point mutation in the Toll-like receptor 4 (TLR4) gene showed decreased NF-kappaB activation and TNFalpha secretion compared with C3H/HeN mouse macrophage when treated with H. pylori. In conclusion, H. pylori-induced NF-kappaB activation in epithelial cells is dependent on cag PAI and contact but does not involve CD14 and IRAK, whereas in macrophage/monocytic cells it is independent of cag PAI or contact but involves CD14 and TLR4.     

Pneumococci induced TLR- and Rac1-dependent NF-kappaB-recruitment to the IL-8 promoter in lung epithelial cells

Streptococcus pneumoniae is the major pathogen of community-acquired pneumonia. The respiratory epithelium constitutes the first line of defense against invading lung pathogens, including pneumococci. We analyzed the involvement of Toll-like receptors (TLR) and Rho-GTPase signaling in the activation of human lung epithelial cells by pneumococci. S. pneumoniae induced release of interleukin-8 (IL-8) by human bronchial epithelial cell line BEAS-2B. Specific inhibition of Rac1 by Nsc23766 or a dominant-negative mutant of Rac1 strongly reduced cytokine release. In addition, pneumococci-related cell activation (IL-8 release, NF-kappaB-activation) depended on MyD88, phosphatidylinositol 3-kinase, and Cdc42 but not on RhoA. Pneumococci enhanced TLR1 and TLR2 mRNA expression in BEAS-2B cells, whereas TLR4 and TLR6 expression was constitutively high. TLR1 and 2 synergistically recognized pneumococci in cotransfection experiments. TLR4, TLR6, LPS-binding protein, and CD14 seem not to be involved in pneumococci-dependent cell activation. At the IL-8 gene promoter, recruitment of phosphorylated NF-kappaB subunit p65 was blocked by inhibition of Rac1, whereas binding of the phosphorylated activator protein-1 subunit c-Jun to the promoter was not diminished. In summary, these results suggest that S. pneumoniae activate human epithelial cells by TLR1/2 and a phosphatidylinositol 3-kinase- and Rac1-dependent NF-kappaB-recruitment to the IL-8 promoter.     

Moraxella catarrhalis lipooligosaccharide selectively upregulates ICAM-1 expression on human monocytes and stimulates adjacent naïve monocytes to produce TNF-alpha through cellular cross-talk

To elucidate the role of Moraxella catarrhalis lipooligosaccharide (LOS) in otitis media with effusion (OME), the effects of LOS on adhesion antigens of human monocytes were investigated. M. catarrhalis LOS selectively enhanced intercellular adhesion molecule 1 (ICAM-1 or CD54) expression on human monocytes by significantly increasing both the surface expression intensity and the percentage of ICAM-1⁺ cells. ICAM-1 upregulation on human monocytes by the LOS required surface CD14, TLR4, NF-κB p65 and c-Jun N-terminal kinase (JNK) activity. Our study also revealed that the LOS-induced surface ICAM-1 expression was partially mediated through a TNF-α dependent autocrine mechanism and could be further augmented by lipopolysaccharide-binding protein in serum. In addition, M. catarrhalis LOS also stimulated human monocytes to produce pro-inflammatory cytokines in both TLR4- and CD14-dependent pathways. Our results also indicated that enhanced surface ICAM-1 expression on monocytes may hinder their adherence to the lung epithelial monolayer. Furthermore, the LOS-activated human monocytes secreted a significantly high level of IL-8, and could stimulate adjacent naïve monocytes to produce TNF-α which was partially mediated via membrane ICAM-1 and IL-8/IL-8RA. These results suggest that M. catarrhalis LOS could induce excessive middle ear inflammation through a cellular cross-talk mechanism during OME.     

Lung NF-kappaB activation and neutrophil recruitment require IL-1 and TNF receptor signaling during pneumococcal pneumonia

Pulmonary inflammation is an essential component of the host defense against Streptococcus pneumoniae infection of the lungs. The early response cytokines, TNF-alpha and IL-1, are rapidly induced upon microbial exposure. Mice deficient in all TNF- and IL-1-dependent signaling receptors were used to determine the roles of these cytokines during pneumococcal pneumonia. The deficiency of signaling receptors for TNF and IL-1 decreased bacterial clearance. Neutrophil recruitment to alveolar air spaces was impaired by receptor deficiency, as was pulmonary expression of the neutrophil chemokines KC and MIP-2. Because NF-kappaB mediates the expression of both chemokines, we assessed NF-kappaB activation in the lungs. During pneumococcal pneumonia, NF-kappaB proteins translocate to the nucleus and activate gene expression; these functions were largely abrogated by the deficiency of receptors for TNF-alpha and IL-1. Thus, the combined deficiency of TNF and IL-1 signaling reduces innate immune responses to S. pneumoniae in the lungs, probably due to essential roles for these receptors in activating NF-kappaB.     

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.     

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.     

Lactoferrin activates macrophages via TLR4-dependent and -independent signaling pathways

Lactoferrin (LF) is a component of innate immunity and is known to interact with accessory molecules involved in the TLR4 pathway, including CD14 and LPS binding protein, suggesting that LF may activate components of the TLR4 pathway. In the present study, we have asked whether bovine LF (bLF)-induced macrophage activation is TLR4-dependent. Both bLF and LPS stimulated IL-6 production and CD40 expression in RAW 264.7 macrophages and in BALB/cJ peritoneal exudate macrophages. However, in macrophages from congenic TLR4(-/-) C.C3-Tlr4(lps-d) mice, CD40 was not expressed while IL-6 secretion was increased relative to wild-type cells. The signaling components NF-kappaB, p38, ERK and JNK were activated in RAW 264.7 cells and BALB/cJ macrophages after bLF or LPS stimulation, demonstrating that the TLR4-dependent bLF activation pathway utilizes signaling components common to LPS activation. In TLR4 deficient macrophages, bLF-induced activation of NF-kappaB, p38, ERK and JNK whereas LPS-induced cell signaling was absent. We conclude from these studies that bLF induces limited and defined macrophage activation and cell signaling events via TLR4-dependent and -independent mechanisms. bLF-induced CD40 expression was TLR4-dependent whereas bLF-induced IL-6 secretion was TLR4-independent, indicating potentially separate pathways for bLF mediated macrophage activation events in innate immunity.     

Viral induction of inflammatory cytokines in human epithelial cells follows a p38 mitogen-activated protein kinase-dependent but NF-kappa B-independent pathway

The initial step in an immune response toward a viral infection is the induction of inflammatory cytokines. This innate immune response is mediated by expression of a variety of cytokines exemplified by TNF-alpha and IL-1beta. A key signal for the recognition of intracellular viral infections is the presence of dsRNA. Viral infections and dsRNA treatment can activate several signaling pathways including the protein kinase R pathway, mitogen-activated protein kinase (MAPK) pathways, and NF-kappaB, which are important in the expression of inflammatory cytokines. We previously reported that activation of protein kinase R was required for dsRNA induction of TNF-alpha, but not for IL-1beta. In this study, we report that activation of the p38 MAPK pathway by respiratory viral infections is necessary for induction of inflammatory cytokines in human bronchial epithelial cells. Inhibition of p38 MAPK by two different pharmacological inhibitors showed that expression of both TNF-alpha and IL-1beta required activation of this signaling pathway. Interestingly, inhibition of NF-kappaB did not significantly reduce viral induction of either cytokine. Our data show that, during the initial infections of epithelial cells with respiratory viruses, activation of the p38 MAPK pathway is associated with induction of inflammation, and NF-kappaB activation may be less important than previously suggested.     

Recombinant HBsAg,an apoptotic-like lipoprotein,interferes with the LPS-induced activation of ERK-1/2 and JNK-1/2 in monocytes

Yeast expressed Hepatitis B surface antigen (rHBsAg) binds to monocytes through interaction with the LPS binding protein (LBP) and the LPS receptor CD14. Charged phospholipids of rHBsAg determine the interaction with these proteins. Although attachment of rHBsAg resembles the pro-inflammatory binding of LPS to CD14, rHBsAg does not activate monocytes and even reduces the expression of pro-inflammatory cytokines by LPS-stimulated monocytes. It is reported here that addition of rHBsAg to LPS-stimulated PBMC often results in increased secretion of IL-10, suggesting a similarity between the interaction of monocytes with apoptotic cells and rHBsAg. Using THP-1 cells, it is shown that IL-10 is not necessary to reduce TNFalpha protein levels. Addition of rHBsAg to LPS-stimulated cells reduces TNFalpha mRNA levels, but does not affect phosphorylation of p65 NF-kappaB and p38 MAP kinase. Instead, a reduced phosphorylation of ERK-1/2 and JNK-1/2 MAP kinases is observed.     

Alternaria induces STAT6-dependent acute airway eosinophilia and epithelial FIZZ1 expression that promotes airway fibrosis and epithelial thickness

The fungal allergen, Alternaria, is specifically associated with severe asthma, including life-threatening exacerbations. To better understand the acute innate airway response to Alternaria, naive wild-type (WT) mice were challenged once intranasally with Alternaria. Naive WT mice developed significant bronchoalveolar lavage eosinophilia following Alternaria challenge when analyzed 24 h later. In contrast to Alternaria, neither Aspergillus nor Candida induced bronchoalveolar lavage eosinophilia. Gene microarray analysis of airway epithelial cell brushings demonstrated that Alternaria-challenged naive WT mice had a >20-fold increase in the level of expression of found in inflammatory zone 1 (FIZZ1/Retnla), a resistin-like molecule. Lung immunostaining confirmed strong airway epithelial FIZZ1 expression as early as 3 h after a single Alternaria challenge that persisted for ≥5 d and was significantly reduced in STAT6-deficient, but not protease-activated receptor 2-deficient mice. Bone marrow chimera studies revealed that STAT6 expressed in lung cells was required for epithelial FIZZ1 expression, whereas STAT6 present in bone marrow-derived cells contributed to airway eosinophilia. Studies investigating which cells in the nonchallenged lung bind FIZZ1 demonstrated that CD45(+)CD11c(+) cells (macrophages and dendritic cells), as well as collagen-1-producing CD45(-) cells (fibroblasts), can bind to FIZZ1. Importantly, direct administration of recombinant FIZZ1 to naive WT mice led to airway eosinophilia, peribronchial fibrosis, and increased thickness of the airway epithelium. Thus, Alternaria induces STAT6-dependent acute airway eosinophilia and epithelial FIZZ1 expression that promotes airway fibrosis and epithelial thickness. This may provide some insight into the uniquely pathogenic aspects of Alternaria-associated asthma.     

Streptococcus pneumoniae induced p38 MAPK- and NF-kappaB-dependent COX-2 expression in human lung epithelium

Streptococcus pneumoniae is a major cause of community-acquired pneumonia and death from infectious diseases in industrialized countries. Lung airway and alveolar epithelial cells comprise an important barrier against airborne pathogens. Cyclooxygenase (COX)-derived prostaglandins, such as PGE(2), are considered to be important regulators of lung function. Herein, we tested the hypothesis that pneumococci induced COX-2-dependent PGE(2) production in pulmonary epithelial cells. Pneumococci-infected human pulmonary epithelial BEAS-2B cells released PGE(2). Expression of COX-2 but not COX-1 was dose and time dependently increased in S. pneumoniae-infected BEAS-2B cells as well as in lungs of mice with pneumococcal pneumonia. S. pneumoniae induced degradation of IkappaBalpha and DNA binding of NF-kappaB. A specific peptide inhibitor of the IkappaBalpha kinase complex blocked pneumococci-induced PGE(2) release and COX-2 expression. In addition, we noted activation of p38 MAPK and JNK in pneumococci-infected BEAS-2B cells. PGE(2) release and COX-2 expression were reduced by p38 MAPK inhibitor SB-202190 but not by JNK inhibitor SP-600125. We analyzed interaction of kinase pathways and NF-kappaB activation: dominant-negative mutants of p38 MAPK isoforms alpha, beta(2), gamma, and delta blocked S. pneumoniae-induced NF-kappaB activation. In addition, recruitment of NF-kappaB subunit p65/RelA and RNA polymerase II to the cox2 promoter depended on p38 MAPK but not on JNK activity. In summary, p38 MAPK- and NF-kappaB-controlled COX-2 expression and subsequent PGE(2) release by lung epithelial cells may contribute significantly to the host response in pneumococcal pneumonia.     

NF-kappaB activation is inhibited in human pulmonary epithelial cells transfected with alpha-melanocyte-stimulating hormone vector

alpha-Melanocyte-stimulating hormone (alpha-MSH) modulates inflammation. We investigated the influence of alpha-MSH on NF-kappaB activation in human pulmonary epithelial cells (A549) using a plasmid vector encoding alpha-MSH (pCMV-ssMSH). Electrophoretic mobility shift assays demonstrated that NF-kappaB activation induced by lipopolysaccharide was inhibited in A549 cells transfected with pCMV-ssMSH. Western blot analysis revealed that this inhibition was linked to preservation of expression of IkappaBalpha protein. Chloramphenicol acetyltransferase assay indicated that NF-kappaB-dependent reporter gene expression was suppressed in A549 cells transfected with pCMV-ssMSH. The findings indicate that anti-inflammatory actions are exerted via modulation of NF-kappaB activation by preservation of IkappaBalpha protein in human pulmonary epithelial cells transfected with alpha-MSH vector. We showed a possibility of gene therapy for chronic inflammatory lung diseases.     

Critical role for T cells in Sephadex-induced airway inflammation: pharmacological and immunological characterization and molecular biomarker identification

Intratracheal instillation of Sephadex particles is a convenient model for assessing the impact of potential anti-inflammatory compounds on lung eosinophilia thought to be a key feature in asthma pathophysiology. However, the underlying cellular and molecular mechanisms involved are poorly understood. We have studied the time course of Sephadex-induced lung eosinophilia, changes in pulmonary T cell numbers, and gene and protein expression as well as the immunological and pharmacological modulation of these inflammatory indices in the Sprague Dawley rat. Sephadex increased T cell numbers (including CD4(+) T cells) and evoked a pulmonary eosinophilia that was associated with an increase in gene/protein expression of the Th2-type cytokines IL-4, IL-5, and IL-13 and eotaxin in lung tissue. Sephadex instillation also induced airway hyperreactivity to acetylcholine and bradykinin. A neutralizing Ab (R73) against the alphabeta-TCR caused 54% depletion of total (CD2(+)) pulmonary T cells accompanied by a significant inhibition of IL-4, IL-13 and eotaxin gene expression together with suppression (65% inhibition) of eosinophils in lung tissue 24 h after Sephadex treatment. Sephadex-induced eosinophilia and Th2 cytokine gene and/or protein expression were sensitive to cyclosporin A and budesonide, compounds that inhibit T cell function, suggesting a pivotal role for T cells in orchestrating Sephadex-induced inflammation in this model.     

Immunoregulatory effects of regulated, lung-targeted expression of IL-10 in vivo

Regulation of pulmonary inflammation involves an intricate balance of both pro- and anti-inflammatory mediators. Acute lung injury can result from direct pulmonary insults that activate alveolar macrophages to respond with increased cytokine expression. Such cytokine gene expression is mediated in part via NF-kappaB. IL-10 has been previously identified as an important endogenous anti-inflammatory cytokine in vivo on the basis of inhibiting NF-kappaB activation; however, the mechanism of this inhibition remains incompletely defined. We hypothesized that IL-10 regulated NF-kappaB activation in vivo via IkappaK inhibition. A bitransgenic mouse that allowed for externally regulated, lung-specific human IL-10 overexpression was generated. In the bitransgenic mice, introduction of doxycycline induced lung-specific, human IL-10 overexpression. Acute induction of IL-10 resulted in significant decreases in bronchoalveolar lavage fluid neutrophils (48%, P = 0.03) and TNF (62%, P < 0.01) following intratracheal LPS compared with bitransgenic negative mice. In vitro kinase assays showed this decrease to correlate to diminished lung IkappaK activity. Furthermore, we also examined the effect of chronic IL-10 overexpression in these transgenic mice. Results show that IL-10 overexpression in lungs of mature mice increased the number of intrapulmonary cells the phenotype of which was skewed toward increased B220+/CD45+ B cells and CD4+ T cells and was associated with increased CC chemokine expression. Thus regulated, lung-specific IL-10 overexpression may have a variety of complex immunologic effects depending on the timing and duration of expression.     

Tumor necrosis factor alpha induction of NF-kappaB requires the novel coactivator SIMPL

A myriad of stimuli including proinflammatory cytokines, viruses, and chemical and mechanical insults activate a kinase complex composed of IkappaB kinase beta (IKK-beta), IKK-alpha, and IKK-gamma/N, leading to changes in NF-kappaB-dependent gene expression. However, it is not clear how the NF-kappaB response is tailored to specific cellular insults. Signaling molecule that interacts with mouse pelle-like kinase (SIMPL) is a signaling component required for tumor necrosis factor alpha (TNF-alpha)-dependent but not interleukin-1-dependent NF-kappaB activation. Herein we demonstrate that nuclear localization of SIMPL is required for type I TNF receptor-induced NF-kappaB activity. SIMPL interacts with nuclear p65 in a TNF-alpha-dependent manner to promote endogenous NF-kappaB-dependent gene expression. The interaction between SIMPL and p65 enhances p65 transactivation activity. These data support a model in which TNF-alpha activation of NF-kappaB dependent-gene expression requires nuclear relocalization of p65 as well as nuclear relocalization of SIMPL, generating a TNF-alpha-specific induction of gene expression.     

CD40 ligation conditions dendritic cell antigen-presenting function through sustained activation of NF-kappaB

An understanding of the biochemical control of dendritic cell (DC) differentiation/activation is essential for improving T cell immunity by various immunotherapeutic approaches, including DC immunization. Ligation of CD40 enhances DC function, including conditioning for CTL priming. NF-kappaB, and particularly RelB, is an essential control pathway for myeloid DC differentiation. Furthermore, RelB regulates B cell Ag-presenting function. We hypothesized that CD40 ligand (CD40L) and TNF-alpha, which differ in their capacity to condition DC, would also differ in their capacity to activate NF-kappaB. DC differentiated for 2 days from monocytes in the presence of GM-CSF and IL-4 were used as a model, as NF-kappaB activity was constitutively low. The capacity of DC to activate T cells following CD40L treatment was enhanced compared with TNF-alpha treatment, and this was NF-kappaB dependent. Whereas RelB/p50 translocation induced by TNF-alpha was attenuated after 6 h, RelB/p50 nuclear translocation induced by CD40L was sustained for at least 24 h. The mechanism of this difference related to enhanced degradation of IkappaBalpha following CD40L stimulation. However, NF-kappaB activation induced by TNF-alpha could be sustained by blocking autocrine IL-10. These data indicate that NF-kappaB activation is essential for T cell activation by DC, and that this function is enhanced if DC NF-kappaB activation is prolonged. Because IL-10 moderates DC NF-kappaB activation by TNF-alpha, sustained NF-kappaB activation can be achieved by blocking IL-10 in the presence of stimuli that induce TNF-alpha.