The Src-Family Kinases Hck and Fgr Regulate Early Lipopolysaccharide-Induced Myeloid Cell Recruitment into the Lung and Their Ability To Secrete Chemokines.

Myeloid leukocyte recruitment into the lung in response to environmental cues represents a key factor for the induction of lung damage. We report that Hck- and Fgr-deficient mice show a profound impairment in early recruitment of neutrophils and monocytes in response to bacterial LPS. The reduction in interstitial and airway neutrophil recruitment was not due to a cell-intrinsic migratory defect, because Hck- and Fgr-deficient neutrophils were attracted to the airways by the chemokine CXCL2 as wild type cells. However, early accumulation of chemokines and TNF-α in the airways was reduced in hck(-/-)fgr(-/-) mice. Considering that chemokine and TNF-α release into the airways was neutrophil independent, as suggested by a comparison between control and neutrophil-depleted mice, we examined LPS-induced chemokine secretion by neutrophils and macrophages in wild type and mutant cells. Notably, mutant neutrophils displayed a marked deficit in their capability to release the chemokines CXCL1, CXCL2, CCL3, and CCL4 and TNF-α in response to LPS. However, intracellular accumulation of these chemokines and TNF-α, as well as secretion of a wide array of cytokines, including IL-1α, IL-1ß, IL-6, and IL-10, by hck(-/-)fgr(-/-) neutrophils was normal. Intriguingly, secretion of CXCL1, CXCL2, CCL2, CCL3, CCL4, RANTES, and TNF-α, but not IL-1α, IL-1ß, IL-6, IL-10, and GM-CSF, was also markedly reduced in bone marrow-derived macrophages. Consistently, the Src kinase inhibitors PP2 and dasatinib reduced chemokine secretion by neutrophils and bone marrow-derived macrophages. These findings identify Src kinases as a critical regulator of chemokine secretion in myeloid leukocytes during lung inflammation.

Selective pulmonary pulsatile perfusion with oxygenated blood during cardiopulmonary bypass attenuates lung tissue inflammation but does not affect circulating cytokine levels.

OBJECTIVE: Improved respiratory outcome has been shown after selective pulsatile pulmonary perfusion (sPPP) during cardiopulmonary bypass (CPB). No contemporary study has analysed the impact of sPPP on alveolar and systemic inflammatory response in humans. METHODS: Sixty-four patients undergoing a coronary artery bypass graft (CABG) were randomized to sPPP or standard CPB (32 patients each). An alveolar-arterial oxygen gradient (A-aDO(2)) was measured preoperatively (T0), at ICU arrival (T1), 3 h postoperatively (T2) and postextubation (T3). The bronchoalveolar lavage (BAL) was collected at T0, T1 and T2. White blood cells (WBCs), neutrophils, mononucleates and lymphocytes in BAL infiltrates were compared between the two groups. A cytokine assay for interleukin-1 (IL-1), IL-8, tumour necrosis factor alpha (TNF-α), monocyte chemotactic protein-1 (MCP-1), growth regulated oncogene-alpha (GRO-α) and interferon (IFN)-γ was collected from the BAL and peripheral blood at the same time-points. Repeated-measure analysis of variance and non-parametric statistics were used to assess the between-group and during time differences. RESULTS: The two groups proved comparable for perioperative variables. A-aDO(2) proved better after sPPP (group-P = 0.0001; group time-P < 0.0001). BAL infiltrates after sPPP showed lower WBCs, neutrophils and lymphocytes (group-P = 0.0001, group time-P = 0.0001 for all) together with higher mononucleates (group-P = 0.0001, group time-P = 0.0001). Proinflammatory cytokines and chemokine MCP-1 were lower in BAL after sPPP (group-P = 0.005, 0.034, 0.036 and 0.005, and group time-P = 0.001, 0.009, 0.001 and 0.0001 for IL-1, IL-8, TNF-α and MCP-1, respectively), whereas the immune modulator IFN-γ significantly augmented after sPPP (time-P = 0.0001) but remained stable after the standard CPB (time-P = 0.101, group-P = 001, group time-P = 0.0001). Indeed, serum cytokines were not different in the two groups during the study (P = NS at single time-points and as a function of time). CONCLUSIONS: sPPP attenuates alveolar inflammation, as demonstrated by the lower neutrophilic/lymphocytic alveolar infiltration, and the secretion of anti-inflammatory rather than proinflammatory mediators.

Modulators of sphingolipid metabolism reduce lung inflammation.

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/ß/γ proteins, and granulocyte chemotactic peptide-2 [GCP-2]), proinflammatory cytokines (IL-1α/ß, 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.

Helicobacter pylori, asthma and allergy.

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-gamma 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.

The neutrophil-activating protein of Helicobacter pylori down-modulates Th2 inflammation in ovalbumin-induced allergic asthma.

The 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.

Toll receptor-mediated regulation of NADPH oxidase in human dendritic cells.

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.

IFN-gamma induces gp91phox expression in human monocytes via protein kinase C-dependent phosphorylation of PU.1.

We previously reported that the stimulation of human blood monocytes with IFN-gamma induces the binding of PU.1 to the gp91(phox) promoter and the consequent expression of gp91(phox). In this study, we show that the effect of IFN-gamma is reproduced by the serine phosphatase inhibitor, okadaic acid, and this suggests that serine kinases could be involved in gp91(phox) expression. We also show that IFN-gamma induces the serine/threonine phosphorylation of PU.1 in cultured monocytes. This phosphorylation, as well as the IFN-gamma-induced PU.1 binding and gp91(phox) protein synthesis, is slightly affected by the casein kinase II inhibitor, daidzein, but is abrogated by the protein kinase C (PKC) -alpha and -beta inhibitor, Go6976, and by synthetic peptides with sequences based on the endogenous pseudosubstrate region of the classical PKC alpha and beta isoforms. In contrast, peptides reproducing the pseudosubstrate region of PKC epsilon were without effect. Moreover, we found that the treatment of monocytes with IFN-gamma induces the nuclear translocation and the activation of PKC alpha and beta I, but not of PKC beta II, and that the IFN-gamma-induced phosphorylation of PU.1 was greatly reduced by LY333531, a selective inhibitor of PKC beta isoforms. Finally, nuclear run-on assays demonstrated that while the PKC inhibitors, Go6976 and LY333531, decrease the IFN-gamma-induced gp91(phox) transcription, the serine phosphatase inhibitor, okadaic acid, enhances the gp91(phox) gene transcription. Our results indicate that in cultured monocytes, IFN-gamma induces the binding of PU.1 to the gp91(phox) promoter and the expression of gp91(phox) by phosphorylation of PU.1 via activation of PKC alpha and/or beta I.

Allergenicity of a hydrolyzed rice infant formula in a guinea pig model.

BACKGROUND: Because sensitization to cow's milk is a common finding in children, the identification of safe alternative protein sources is important in the management of childhood allergy. OBJECTIVE: To evaluate, in an animal model, the allergenicity of a novel formula based on hydrolyzed rice proteins. METHODS: We conducted an experiment involving 130 guinea pigs, from 7 to 12 days old at the onset of the study. The animals were divided into 13 groups and were given, ad libitum, one of the following liquids to drink: (1) rice hydrolysate formula (RF), (2) a conventional cow's milk formula (CMF), or (3) water. After a 37-day sensitization period, a challenge was given, consisting of an intravenous injection of either isolated proteins or ultracentrifuged formulas (uCMF and uRF). Specific IgG antibodies to beta-lactoglobulin, casein, and whole rice protein were measured by enzyme-linked immunosorbent assay. RESULTS: When animals fed CMF were challenged with beta-lactoglobulin, casein, or whole uCMF, they showed significantly more reactions than did those fed RF when challenged with the same proteins (P < 0.001). In the groups fed RF, no reaction was observed after challenge with uRF, and only 2 mild reactions occurred after challenge with rice protein. Very low levels of specific IgG antibodies to rice protein were noted in all the groups, including the RF-fed animals, and no significant differences were evident between groups. CONCLUSIONS: The findings suggest that this new formula based on hydrolyzed rice proteins has a very low sensitizing capability.

Fgr deficiency results in defective eosinophil recruitment to the lung during allergic airway inflammation.

Using a mouse model of allergic lung inflammation, we found that mice deficient of Fgr, a Src family tyrosine kinase highly expressed in myelomonocytic cells, fail to develop lung eosinophilia in response to repeated challenge with aerosolized OVA. Both tissue and airway eosinophilia were markedly reduced in fgr(-/-) mice, whereas mice with the sole deficiency of Hck, another Src family member, responded normally. Release of allergic mediators, such as histamine, IL-4, RANTES/CCL5, and eotaxin/CCL11, in the airways of OVA-treated animals was equal in wild-type and fgr(-/-) mice. However, lung eosinophilia in Fgr-deficient mice correlated with a defective accumulation of GM-CSF and IL-5 in the airways, whereas secretion of these cytokines by spleen cells in response to OVA was normal. Examination of mRNA expression in whole lung tissue allowed us to detect comparable expression of transcripts for eotaxin/CCL11, macrophage-inflammatory protein-1 alpha/CCL3, macrophage-inflammatory protein-1 beta/CCL4, monocyte chemoattractant protein-1/CCL2, TCA-3/CCL1, IL-4, IL-10, IL-2, IL-3, IL-9, IL-15, and IFN-gamma in OVA-sensitized wild-type and fgr(-/-) mice. In contrast, the increase in IL-5 and IL-13 mRNA expression was lower in fgr(-/-) compared with wild-type mice. These findings suggest that deficiency of Fgr results in a marked reduction of lung eosinophilia and the establishment of a positive feedback loop based on autocrine secretion of eosinophil-active cytokines. These results identify Fgr as a novel pharmacological target to control allergic inflammation.