Activation of NADPH oxidase represents an essential mechanism of defense against pathogens. Dendritic cells (DC) are phagocytic cells specialized in Ag presentation rather than in bacteria killing. Human monocyte-derived DC were found to express the NADPH oxidase components and to release superoxide anions in response to phorbol esters and phagocytic agonists. The NADPH oxidase components p47phox and gp91phox were down-regulated during monocyte differentiation to DC, and maturation of DC with pathogen-derived molecules, known to activate TLRs, increased p47phox and gp91phox expression and enhanced superoxide anions release. Similar results were obtained with plasmacytoid DC following maturation with influenza virus. In contrast, activation of DC by immune stimuli (CD40 ligand) did not regulate NADPH oxidase components or respiratory burst. NADPH oxidase-derived oxygen radicals did not play any role in DC differentiation, maturation, cytokine production, and induction of T cell proliferation, as based on the normal function of DC generated from chronic granulomatous disease patients and the use of an oxygen radical scavenger. However, NADPH oxidase activation was required for DC killing of intracellular Escherichia coli. It is likely that the selective regulation of oxygen radicals production by pathogen-activated DC may function to limit pathogen dissemination during DC trafficking to secondary lymphoid tissues.

SummaryThe Helicobacter pylori neutrophil-activating protein (HP-NAP) is able in vitro to elicit IL-12 and IL-23 production via agonistic interaction with toll-like receptor 2, and to promote Th1 polarization of allergen-specific T-cell responses. This study was aimed to assess whether systemic/intraperitoneal and/or mucosal HP-NAP administration inhibited the Th2-mediated bronchial inflammation using a mouse model of allergic asthma induced by inhaled ovalbumin (OVA). Systemic HP-NAP delivery markedly reduced the lung eosinophilia in response to repeated challenge with aerosolized OVA. Likewise, the production of IL-4, IL-5 and GM-CSF was significantly lower in the bronchoalveolar lavage of animals treated with systemic HP-NAP plus OVA than that of animals treated with OVA alone. Systemic HP-NAP also significantly resulted in both reduction of total serum IgE and increase of IL-12 plasma levels. Mucosal administration of HP-NAP was equally successful as the systemic delivery in reducing eosinophilia, IgE and Th2 cytokine levels in bronchoalveolar lavage. However, no suppression of lung eosinophilia and bronchial Th2 cytokines was observed in toll-like receptor 2-knock-out mice following HP-NAP treatment. These results identify HP-NAP as a candidate for novel strategies of prevention and treatment of allergic diseases.

The investigation of novel targets for the treatment of cystic fibrosis (CF) lung inflammation is a major priority, considering that no effective therapy is available for this purpose. Consistent with the evidence that the sphingolipid (SL) ceramide regulates airway inflammation and infection in mice and patients with CF, SLs were identified as targets for treating pulmonary disorders, including CF. Because miglustat, an inhibitor of the synthesis of glycosphingolipids, reduces the Pseudomonas aeruginosa-dependent transcription of the IL-8 gene in bronchial cells, we examined the effects of miglustat and amitriptyline, another drug affecting ceramide metabolism, on the expression of 92 genes implicated in host immune defense. Infection with the P. aeruginosa strain PAO1 up-modulated the expression of 14 (27%) genes in IB3-1 cells and 15 (29%) genes in CF primary respiratory epithelia grown at an air-liquid interface, including chemokines (IL-8, growth-regulated Gro-α/β/γ proteins, and granulocyte chemotactic peptide-2 [GCP-2]), proinflammatory cytokines (IL-1α/β, IL-6, and TNF-α), and the intercellular adhesion molecule-1, nuclear factor kB1, toll like receptor 2, and human defensin B4 genes, confirming that bronchial epithelium is an important source of inflammatory mediators. Both miglustat and amitriptyline reduced the immune response, an effect that paralleled a decrease in the P. aeruginosa-induced accumulation of ceramide. Miglustat (100 mg/kg), given to C57BL/6 mice once daily for a period of 3 consecutive days before lipopolysaccharide (LPS) challenge, strongly reduced the number of neutrophils recruited in the airways and the expression of the keratinocyte-derived chemokine in lung extracts. Collectively, these results indicate that targeting the metabolism of SLs can down-modulate the recruitment of neutrophils into the lung.

Human blood monocytes lose their capability to produce microbicidal oxidants during culture. We report that this process is associated with decreased gp91phox, p22phox and p47phox expression, release of PU.1 and CP‐1 from gp91phox promoter, and PU.1 from p47phox promoter. However, in presence of IFN‐γ or TNF‐α, the superoxide anion (O 2–) production, the p47phox, gp91phox and p22phox expression, and the binding of PU.1 and CP‐1 to DNA are maintained at the high levels observed in blood monocytes. To clarify the role of PU.1 in the expression of NADPH oxidase components, oligonucleotides competing for PU.1‐DNA binding were added to cultured monocytes. These oligonucleotides abrogated the maintenance of gp91phox and p22phox expression by IFN‐γ and TNF‐α, but did not inhibit the effect of these cytokines on p47phox expression and O 2– production. Our results indicate that in monocytes the IFN‐γ‐ and TNF‐α‐induced expression of gp91phox and p22phox, but not p47phox, requires the binding of PU.1 to gp91phox promoter. However, the preservation of O 2– production by IFN‐γ and TNF‐α is unrelated to their effect on gp91phox and p22phox expression.

The relationship between nitric oxide in exhaled air, levels of sputum eosinophils, sputum eosinophil cationic protein (ECP) and urinary eosinophil protein X (EPX) excretion has not yet been investigated in corticosteroid-dependent childhood asthma.Therefore, taking 25 children with stable asthma (mean age 11.2 yrs) treated with inhaled corticosteroids and nine nonatopic healthy control children (mean age 12.8 yrs) the level of exhaled NO was measured by means of a chemiluminescence analyser before and after sputum induction. This was conducted as a slow vital capacity manoeuvre under standardized conditions with a target flow of 70 mL . s -1 against a resistance of 100 cmH 2 O . L -1 . s. Sputum induction was performed by inhalation of hypertonic saline (3, 4, and 5%) in a standardized manner and a single sample of urine was collected.Exhaled NO (p=0.01), absolute eosinophil cell counts in sputum (p=0.02), sputum ECP (p=0.09) and urinary EPX excretion (p=0.02) were higher in asthmatics compared to control children. Exhaled NO was positively correlated with sputum ECP (r s =0.59, p=0.002), urinary EPX (r s =0.42, p=0.03), and sputum eosinophils (r s =0.30, p=0.15) in the asthmatic children. These correlations appeared to be pronounced after sputum induction, where NO values had decreased (p=0.01). None of the correlations were observed in the group of nonatopic control subjects. Additionally there were significant correlations between sputum ECP and sputum eosinophils (r s =0.69, p< 0.001) as well as between sputum ECP and urinary EPX excretion (r s =0.58, p=0.003) in the asthmatics.Exhaled NO provides information about the degree of eosinophilic airway inflammation and thus appears to be a useful and easy-to-perform inflammatory marker in corticosteroid-dependent asthma. Eur Respir J 1999; 13: 1391±1395.

Exposure to house dust mite antigen can induce airway epithelial shedding even in subjects with low eosinophil airway infiltration, thus supporting the idea that epithelial damage in asthmatics sensitized to Dermatophagoides may be due to a proteolytic activity of the mite major antigens.

Bronchial asthma and allergic diseases are orchestrated by T-cells producing T-helper type 2 (Th2) cytokines, such as interleukin-4 (IL-4) and IL-5, and are inhibited by Th1 responses. Helicobacter pylori has chronically infected the human population for c. 100 000 years and preferentially elicits a Th1 mucosal immune response with the production of interferon-g and IL-12. Among several bacterial factors, the neutrophil-activating protein of H. pylori (HP-NAP) not only plays a key role in driving Th1 inflammation but it is also able to inhibit Th2 responses in vitro and in vivo in allergic bronchial asthma, in humans and mice. Both systemic and mucosal administrations of HP-NAP are successful in reducing eosinophilia, immunoglobulin E and systemic Th2 cytokines at the bronchial level. Thus, these results identify HP-NAP as a candidate for novel strategies for the prevention and treatment of allergic diseases.

We previously reported that the stimulation of human blood monocytes with IFN-γ induces the binding of PU.1 to the gp91phox promoter and the consequent expression of gp91phox. In this study, we show that the effect of IFN-γ is reproduced by the serine phosphatase inhibitor, okadaic acid, and this suggests that serine kinases could be involved in gp91phox expression. We also show that IFN-γ induces the serine/threonine phosphorylation of PU.1 in cultured monocytes. This phosphorylation, as well as the IFN-γ-induced PU.1 binding and gp91phox protein synthesis, is slightly affected by the casein kinase II inhibitor, daidzein, but is abrogated by the protein kinase C (PKC) -α and -β inhibitor, Go6976, and by synthetic peptides with sequences based on the endogenous pseudosubstrate region of the classical PKC α and β isoforms. In contrast, peptides reproducing the pseudosubstrate region of PKC ε were without effect. Moreover, we found that the treatment of monocytes with IFN-γ induces the nuclear translocation and the activation of PKC α and βI, but not of PKC βII, and that the IFN-γ-induced phosphorylation of PU.1 was greatly reduced by LY333531, a selective inhibitor of PKC β isoforms. Finally, nuclear run-on assays demonstrated that while the PKC inhibitors, Go6976 and LY333531, decrease the IFN-γ-induced gp91phox transcription, the serine phosphatase inhibitor, okadaic acid, enhances the gp91phox gene transcription. Our results indicate that in cultured monocytes, IFN-γ induces the binding of PU.1 to the gp91phox promoter and the expression of gp91phox by phosphorylation of PU.1 via activation of PKC α and/or βI.