Asthma phenotypes and IgE responses.

The discovery of IgE represented a major breakthrough in allergy and asthma research, whereas the clinical interest given to IgE in asthma has been blurred until the arrival of anti-IgE biotherapy. Novel facets of the complex link between IgE and asthma have been highlighted by the effect of this treatment and by basic research. In parallel, asthma phenotyping recently evolved to the concept of endotypes, relying on identified/suspected pathobiological mechanisms to phenotype patients, but has not yet clearly positioned IgE among biomarkers of asthma.In this review, we first summarise recent knowledge about the regulation of IgE production and its main receptor, FcεRI. In addition to allergens acting as classical IgE inducers, viral infections as well as air pollution may trigger the IgE pathway, notably resetting the threshold of IgE sensitivity by regulating FcεRI expression. We then analyse the place of IgE in different asthma endo/phenotypes and discuss the potential interest of IgE among biomarkers in asthma.

Diagnostic Accuracy and Safety of CT-Guided Percutaneous Transthoracic Needle Biopsies: 14-Gauge versus 22-Gauge Needles.

PURPOSE: To compare the diagnostic accuracy and safety of a 14-gauge core needle versus a 22-gauge fine needle in the evaluation of thoracic lesions by CT-guided percutaneous transthoracic needle biopsy (TTNB). MATERIALS AND METHODS: Medical charts of all patients who underwent CT-guided percutaneous transthoracic core-needle biopsies (CNBs) with a 14-gauge Spirotome device (99 patients, 102 procedures) and fine-needle biopsies (FNBs) with a 22-gauge Rotex needle (92 patients, 102 procedures) between 2007 and 2013 at a single academic institution were retrospectively reviewed. Variables that could influence diagnostic accuracy and safety were collected. RESULTS: The overall and cancer-specific diagnostic accuracy rates were 90% and 94%, respectively, with CNB, versus 82% and 89% with FNB. Precise cancer type/subtype was provided by 97% of CNBs versus 65% of FNBs (P < .001). In patients with lung cancer considered for targeted therapy, biomarker analyses were feasible in 80% of CNBs versus 0% of FNBs (P < .001). The rate of pneumothorax was significantly higher with CNB versus FNB (31% vs 19%; P = .004), but chest tube insertion rates were similar (10% vs 11%, respectively). Major bleeding complications occurred in 1% of CNBs versus 2% of FNBs and were associated with one death in the CNB group. CONCLUSIONS: Percutaneous transthoracic CNB with a 14-gauge Spirotome needle provided better characterization of cancer lesions and allowed biomarker analyses without a significant increase in major procedural complications.

Polymeric immunoglobulin receptor down-regulation in chronic obstructive pulmonary disease. Persistence in the cultured epithelium and role of transforming growth factor-ß.

RATIONALE: The generation of protective secretory IgA relies on the epithelial polymeric immunoglobulin receptor (pIgR). pIgR expression is reduced in chronic obstructive pulmonary disease (COPD), but correlation to disease severity and underlying mechanisms remains unknown. OBJECTIVES: To address the hypothesis that pIgR down-regulation in COPD concerns severe disease in relation to aberrant programming of the bronchial epithelium. METHODS: Surgical lung tissue and primary bronchial epithelium (cultured in air-liquid interface, ALI) obtained from a large series of patients (n = 116) were studied for pIgR expression and regulation. MEASUREMENTS AND MAIN RESULTS: pIgR immunostaining in the bronchial epithelium is decreased in severe COPD. In contrast, pIgR transcription was up-regulated in smokers with or without COPD. In ALI (vs. submerged) cultures, pIgR expression was strongly induced, whereas pIgR expression and IgA-transcytosis capacity were decreased in cultures from subjects with severe COPD as compared with control subjects. In addition, COPD cultures released more transforming growth factor-ß1 (TGF-ß1), reflecting increased epithelial TGF-ß1 immunostaining in COPD lung tissue. Finally, besides inducing epithelial dedifferentiation, exogenous TGF-ß1 dose-dependently inhibited pIgR production, whereas pIgR increased on blockade of TGF-ß1 activity during ALI differentiation. CONCLUSIONS: pIgR down-regulation in COPD correlates with disease severity, and the bronchial epithelium reconstituted in vitro from these patients retains its aberrant imprinting for pIgR expression. This study also links pIgR down-regulation to TGF-ß-driven reprogramming of the bronchial epithelium, which results in impaired lung IgA immunity in patients with COPD.

Tyrosine kinase inhibitors in pulmonary arterial hypertension: a double-edge sword?

New treatments for pulmonary arterial hypertension (PAH) are a crucial need. The increased proliferation, migration, and survival of pulmonary vascular cells within the pulmonary artery wall in PAH have allowed successful transposition of pathophysiological elements from oncologic researches. Next steps will require translation of these biological advances in PAH therapeutic arsenal and guidelines. This review synthesizes recent data concerning the role of receptor tyrosine kinases and their inhibitors in PAH, with implications in animal models and humans. Results of clinical trials are now accumulating to establish beneficial role of tyrosine kinase inhibitors (TKIs) in PAH and further findings are expected in the near future. Beside this curative approach, evidences of a possible TKI-induced cardiotoxicity are emerging. These safety issues raise concern about a potential amplified harmful effect in PAH, a pathology characterized by an underlying cardiac dysfunction. In addition, analyses of PAH registries shed light on a selective pulmonary vascular toxicity triggered by TKIs, especially dasatinib. These possible dual effects of the TKIs in PAH need to be taken in account for future pharmacological development of this therapeutic class in PAH.

Loss of polymeric immunoglobulin receptor expression is associated with lung tumourigenesis.

Polymeric immunoglobulin receptor (pIgR) expression is downregulated in lung cancer, but its implications in lung tumourigenesis remain unknown. We hypothesised that loss of pIgR expression occurs early, and is associated with cell proliferation and poor prognosis. pIgR expression was evaluated by immunohistochemistry in airways of patients with normal mucosa, pre-invasive lesions and invasive lesions, and correlated with clinical outcomes. 16-HBE and A549 cells stably transfected with pIgR were tested for proliferation, apoptosis and cell cycle progression. Immunostaining was strong in normal epithelium, but severely reduced in pre-invasive lesions and most lung cancers. Persistent expression was associated with younger age and adenocarcinoma subtype but not survival. pIgR overexpression significantly reduced A549 and 16-HBE proliferation. Growth inhibition was not due to cell cycle arrest, increased apoptosis or endoplasmic reticulum stress, but we observed altered expression of genes encoding for membrane proteins, including NOTCH3. Interestingly, NOTCH3 expression was inversely correlated with pIgR expression in cell lines and tissues. pIgR expression was lost in most lung cancers and pre-invasive bronchial lesions, suggesting that pIgR downregulation is an early event in lung tumourigenesis. pIgR overexpression in A549 and 16-HBE cells inhibited proliferation. Future investigations are required to determine the mechanisms by which pIgR contributes to cell proliferation.

Induction of interleukin-10 expression through Fcalpha receptor in human monocytes and monocyte-derived dendritic cells: role of p38 MAPKinase.

As previously reported by others for immunoglobulin (Ig)G, we observed that IgA can induce interleukin (IL)-10 expression in human monocytes. In this study, we explored the molecular mechanisms of IL-10 induction by IgA in monocytes and monocyte-derived dendritic cells (MD-DCs). Monomeric IgA induced IL-10 production in monocytes and this production was further increased upon IgA cross-linking. Similar IL-10 responses were observed in monocytes and autologous MD-DCs, and were inhibited (by approximately 77%) by preincubation with a blocking mAb to FcalphaRI. IL-10 induction by IgA correlated with activation of MAPKinases ERK1/2, p38 and JNK, whereas only p38-inhibitor SB-203580 inhibited IL-10 induction. Upon IgA stimulation, AP-1, NFkappaB and Sp1 transcription factors were activated and inhibitors of NFkappaB and of Sp1 suppressed IgA-driven transcriptional activation of IL-10. In addition, p38 MAPK activation appeared that it was required to control nuclear translocation of NFkappaB and Sp1 upon IgA stimulation. Therefore, in human monocytes and MD-DCs the mechanisms of IL-10 induction by IgA involve p38 MAPK-dependent recruitment of both NFkappaB and Sp1.

Dual effect of neutrophils on pIgR/secretory component in human bronchial epithelial cells: role of TGF-beta.

Neutrophils have a dual affect on epithelial pIgR/SC, the critical receptor for transcellular routing of mucosal IgA, but mechanisms of pIgR/SC upregulation remain elusive. Requirements of cytokine, redox, and signalling pathways for pIgR/SC production were assessed in human bronchial epithelial (Calu-3) cells cocultured with increasing numbers of blood neutrophils. Increased SC production was observed after incubation for 48 hrs with intermediate neutrophil numbers (1.25 to 2.5 x 10(6)), was favoured by the elastase inhibitor SLPI, and correlated with increased TGF-beta production. Exogenous TGF-beta stimulated SC production with a maximal effect at 48 hrs and both TGF-beta- and neutrophil-driven SC upregulation were dependent on redox balance and p38 MAP-kinase activation. This paper shows that activated neutrophils could upregulate epithelial pIgR/SC production through TGF-beta-mediated activation of a redox-sensitive and p38 MAPK-dependent pathway. An imbalance between the two neutrophil-driven opposite mechanisms (SC upregulation and SC degradation) could lead to downregulation of pIgR/SC, as observed in severe COPD.

Therapeutic Potential of Focal Adhesion Kinase Inhibition in Small Cell Lung Cancer.

Small cell lung cancer (SCLC) has a poor prognosis. Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase regulating cell proliferation, survival, migration, and invasion, which is overexpressed and/or activated in several cancers, including SCLC. We wanted to determine whether FAK contributes to SCLC aggressive behavior. We first evaluated the effect of FAK small-molecule inhibitor PF-573,228 in NCI-H82, NCI-H146, NCI-H196, and NCI-H446 SCLC cell lines. PF-573,228 (0.1-5 µmol/L) inhibited FAK activity by decreasing phospho-FAK (Tyr397), without modifying total FAK expression. PF-573,228 decreased proliferation, decreased DNA synthesis, induced cell-cycle arrest in G2-M phases, and increased apoptosis in all cell lines. PF-573,228 also decreased motility in adherent cell lines. To make sure that these effects were not off-target, we then used a genetic method to inhibit FAK in NCI-H82 and NCI-H446, namely stable transduction with FAK shRNA and/or FAK-related nonkinase (FRNK), a splice variant lacking the N-terminal and kinase domains. Although FAK shRNA transduction decreased total and phospho-FAK (Tyr397) expression, it did not affect proliferation, DNA synthesis, or progression through cell cycle. However, restoration of FAK-targeting (FAT) domain (attached to focal adhesion complex where it inhibits pro-proliferative proteins such as Rac-1) by FRNK transduction inhibited proliferation, DNA synthesis, and induced apoptosis. Moreover, although FAK shRNA transduction increased active Rac1 level, FRNK reexpression in cells previously transduced with FAK shRNA decreased it. Therefore, FAK appears important in SCLC biology and targeting its kinase domain may have a therapeutic potential, while targeting its FAT domain should be avoided to prevent Rac1-mediated protumoral activity.

Increased Expression and Activation of FAK in Small-Cell Lung Cancer Compared to Non-Small-Cell Lung Cancer.

INTRODUCTION: Focal adhesion kinase (FAK) plays a crucial role in cancer development and progression. FAK is overexpressed and/or activated and associated with poor prognosis in various malignancies. However, in lung cancer, activated FAK expression and its prognostic value are unknown. METHODS: FAK and activated FAK (phospho-FAK Y397) expressions were analyzed by multiplex immunofluorescence staining in formalin-fixed paraffin-embedded tissues from 95 non-small-cell lung cancer (NSCLC) and 105 small-cell lung cancer (SCLC) patients, and 37 healthy donors. The FAK staining score was defined as the percentage (%) of FAK-stained tumor area multiplied by (×) FAK mean intensity and phospho-FAK staining score as the (% of phospho-FAK-stained area of low intensity × 1) + (% of phospho-FAK-stained area of medium intensity × 2) + (% of the phospho-FAK-stained area of high intensity × 3). FAK and phospho-FAK staining scores were compared between normal, NSCLC, and SCLC tissues. They were also tested for correlations with patient characteristics and clinical outcomes. RESULTS: The median follow-up time after the first treatment was 42.5 months and 6.4 months for NSCLC and SCLC patients, respectively. FAK and phospho-FAK staining scores were significantly higher in lung cancer than in normal lung and significantly higher in SCLC compared to NSCLC tissues (p < 0.01). Moreover, the ratio between phospho-FAK and FAK staining scores was significantly higher in SCLC than in NSCLC tissues (p < 0.01). However, FAK and activated FAK expression in lung cancer did not correlate with recurrence-free and overall survival in NSCLC and SCLC patients. CONCLUSIONS: Total FAK and activated FAK expressions are significantly higher in lung cancer than in normal lung, and significantly higher in SCLC compared to NSCLC, but are not prognostic biomarkers in this study.

LPS induces IL-10 production by human alveolar macrophages via MAPKinases- and Sp1-dependent mechanisms.

BACKGROUND: IL-10 is a cytokine mainly produced by macrophages that plays key roles in tolerance to inhaled antigens and in lung homeostasis. Its regulation in alveolar macrophages (HAM), the resident lung phagocytes, remains however unknown. METHODS: The present study investigated the role of intracellular signalling and transcription factors controlling the production of IL-10 in LPS-activated HAM from normal nonsmoking volunteers. RESULTS: LPS (1-1000 pg/ml) induced in vitro IL-10 production by HAM, both at mRNA and protein levels. LPS also activated the phosphorylation of ERK, p38 and JNK MAPkinases (immunoblots) and Sp-1 nuclear activity (EMSA). Selective inhibitors of MAPKinases (respectively PD98059, SB203580 and SP600125) and of Sp-1 signaling (mithramycin) decreased IL-10 expression in HAM. In addition, whilst not affecting IL-10 mRNA degradation, the three MAPKinase inhibitors completely abolished Sp-1 activation by LPS in HAM. CONCLUSION: These results demonstrate for the first time that expression of IL-10 in lung macrophages stimulated by LPS depends on the concomitant activation of ERK, p38 and JNK MAPKinases, which control downstream signalling to Sp-1 transcription factor. This study further points to Sp-1 as a key signalling pathway for IL-10 expression in the lung.

Phagocytosis and killing of intracellular pathogens: interaction between cytokines and antibiotics.

Phagocytosis and bacterial killing are the primary functions of macrophages. Among the mechanisms involved in the phagocytic process, cytokines, especially those of T-helper 1 profile, appear to influence considerably the internalization and the intracellular fate of the pathogen within the macrophage. In particular, the evidence for a cooperation of cytokines with antibiotics in intracellular infection could provide new therapeutic approaches to intracellular infectious diseases in the future.

Discovery of new membrane-associated proteins overexpressed in small-cell lung cancer.

INTRODUCTION: Small-cell lung cancer (SCLC) is the most aggressive subtype of lung cancer, with no early detection strategy or targeted therapy currently available. We hypothesized that difference gel electrophoresis (DIGE) may identify membrane-associated proteins (MAPs) specific to SCLC, advance our understanding of SCLC biology, and discover new biomarkers of SCLC. METHODS: MAP lysates were prepared from three SCLCs, three non-small-cell lung cancers, and three immortalized normal bronchial epithelial cell lines and coanalyzed by DIGE. Subsequent protein identification was performed by mass spectrometry. Proteins were submitted to Ingenuity Pathway Analysis. Candidate biomarkers were validated by Western blotting (WB) and immunohistochemistry (IHC). RESULTS: Principal component analysis on the global DIGE data set demonstrated that the four replicates derived from each of the nine cell lines clustered closely, as did samples within the same histological group. One hundred thirty-seven proteins were differentially expressed in SCLC compared with non-small-cell lung cancer and immortalized normal bronchial epithelial cells. These proteins were overrepresented in cellular/tissue morphology networks. Dihydropyrimidinase-related protein 2, guanine nucleotide-binding protein alpha-q, laminin receptor 1, pontin, and stathmin 1 were selected as candidate biomarkers among MAPs overexpressed in SCLC. Overexpression of all candidates but RSSA in SCLC was verified by WB and/or IHC on tissue microarrays. These proteins were significantly associated with SCLC histology and survival in univariables analyses. CONCLUSION: DIGE analysis of a membrane-associated subproteome discovered overexpression of dihydropyrimidinase-related protein 2, guanine nucleotide-binding protein alpha-q, RUVB1, and stathmin 1 in SCLC. Results were verified by WB and/or IHC in primary tumors, suggesting that investigating their functional relevance in SCLC progression is warranted. Association with survival requires further validation in larger clinical data sets.

Mucosal immunity in asthma and chronic obstructive pulmonary disease: a role for immunoglobulin A?

Despite our knowledge on the role of IgA in mucosal homeostasis and host defense and clinical evidence suggesting deficient first-line defense mechanisms in chronic airway disorders, little is known regarding its role in asthma and chronic obstructive pulmonary disease (COPD). Studies suggest that the mucosal IgA response is impaired in COPD, and a deficient transport of IgA across the bronchial epithelium in COPD has been identified, possibly involving neutrophil proteinases, which may degrade the Ig receptor mediating this transepithelial routing. In contrast, the IgA response to allergens in patients with asthma may play a pathogenic role through eosinophil activation. Thus, secretory IgA can induce eosinophil degranulation in vitro, a feature in keeping with the correlations observed in vivo between airway IgA levels and eosinophil cationic protein during late asthmatic responses. Selective IgA deficiency is associated with an increased prevalence of atopy, and a protective role of IgA has been seen in murine models of asthma, delineating the complexity of the IgA system in the airway mucosa. Future studies will hopefully yield better knowledge of IgA biology and lung mucosal immunity and help to use more efficiently the mucosal route for immunotherapy or target specific genes in inflamed airways.

Effect of IgA on respiratory burst and cytokine release by human alveolar macrophages: role of ERK1/2 mitogen-activated protein kinases and NF-kappaB.

Human alveolar macrophages (HAM) express FcalphaR receptors for immunoglobulin (Ig)A which could link humoral and cellular branches of lung immunity. Here, we investigate the effects of polymeric (p-IgA) and secretory (S-IgA) IgA interaction with Fc(alpha)R on lipopolysaccharide (LPS)- and phorbol myristate acetate (PMA)-activated respiratory burst and TNF-alpha release by HAM. Activation of HAM with LPS and PMA increases the respiratory burst and TNF-alpha release through activation of the extracellular signal-related protein kinases 1 and 2 (ERK1/2) pathway, because these effects are inhibited by treatment of HAM with PD98059, a selective inhibitor of mitogen-activated protein (MAP)/ERK kinases (MEK) pathway. S-IgA and p-IgA downregulate the LPS-increased respiratory burst in HAM through an inhibition of ERK1/2 activity. In contrast, p- and S-IgA induce an increase in the respiratory burst of PMA-treated HAM. This effect is associated with an upregulation by IgA of the PMA-induced phosphorylation of ERK1/2 and is also inhibited by PD98059. Moreover, p-IgA and S-IgA enhance TNF-alpha release by HAM through an alternative pathway distinct from ERK1/2. Because LPS is known to activate nuclear factor-kappaB (NF-kappaB) in HAM, we evaluate the effect of IgA on NF-kappaB. Treatment of HAM with LPS, p- and S-IgA, but not PMA, induces NF-kappaB activation through IkappaBalpha phosphorylation and subsequent proteolysis. Antioxidants, namely N-acetylcysteine (NAC) and glutathione (GSH), have no effects on IgA-mediated NF-kappaB nuclear translocation and only a minor and late effect on that of LPS, suggesting that reactive oxygen intermediates (ROI) play a minor role in HAM activation through NF-kappaB. TNF-alpha release by LPS-activated HAM is sensitive to NF-kappaB inhibition and only partly to oxidant scavenging. In contrast, TNF-alpha release by IgA-treated HAM is not dependent on oxidants and only partly dependent on NF-kappaB. Our results show a differential HAM regulation by IgA through both dependent and independent modulation of ERK pathway. In addition, IgA activates NF-kappaB and this effect was independent on oxidants. These data may help to understand the role of IgA in both lung protection and inflammation.

Secretory component is cleaved by neutrophil serine proteinases but its epithelial production is increased by neutrophils through NF-kappa B- and p38 mitogen-activated protein kinase-dependent mechanisms.

We previously showed that expression of polymeric immunoglobulin receptor (pIgR)/secretory component (SC), the epithelial receptor assuming transport of polymeric IgA in mucosal secretions, is strongly decreased in severe chronic obstructive pulmonary disease. Here, we evaluated in vitro the effects of polymorphonuclear neutrophil (PMN) mediators on pIgR/SC. On polyacrylamide gel electrophoresis analysis, soluble SC was rapidly cleaved by supernatants from phorbol-myristate-acetate-activated PMN, through a serine proteinase activity. Moreover, purified PMN serine proteinases also cleaved SC. Similarly, polymeric IgA was rapidly cleaved in monomers by neutrophil elastase, whereas secretory immunoglobulin A was relatively resistant to neutrophil elastase. Surface pIgR on human bronchial epithelial cells was also cleaved by serine proteinases, as shown by immunofluorescence. In contrast, pIgR/SC production by cultured epithelial cells (quantified by enzyme-linked immunosorbent assay) was significantly increased by supernatants from interleukin-8/formylmethionylleucylphenylalanine-activated PMN (122.6 +/- 17.3 versus 70.9 +/- 9 ng/mg protein, P < 0.01). Upregulation of pIgR/SC production by bronchial epithelial cells was abolished by nuclear factor kappa B- and p38 mitogen-activated protein kinase (MAPK) inhibitors. Moreover, supernatants from interleukin-8/formylmethionylleucylphenylalanine-activated PMN induced the phosphorylation of I kappa B-alpha and p38 MAPK in epithelial cells, independently of serine proteinases. Thus, PMN serine proteinases cleave pIgR/SC, whereas activated PMN induce an increased pIgR/SC expression through epithelial activation of nuclear factor kappa B and p38 MAPK pathways.

IL-9 inhibits oxidative burst and TNF-alpha release in lipopolysaccharide-stimulated human monocytes through TGF-beta.

IL-9 is a Th2 cytokine that exerts pleiotropic activities on T cells, B cells, mast cells, hematopoietic progenitors, and lung epithelial cells, but no effect of this cytokine has been reported so far on mononuclear phagocytes. Human blood monocytes preincubated with IL-9 for 24 h before LPS or PMA stimulation exhibited a decreased oxidative burst, even in the presence of IFN-gamma. The inhibitory effect of IL-9 was specifically abolished by anti-hIL-9R mAb, and the presence of IL-9 receptors was demonstrated on human blood monocytes by FACS. IL-9 also down-regulated TNF-alpha and IL-10 release by LPS-stimulated monocytes. In addition, IL-9 strongly up-regulated the production of TGF-beta1 by LPS-stimulated monocytes. The suppressive effect of IL-9 on the respiratory burst and TNF-alpha production in LPS-stimulated monocytes was significantly inhibited by anti-TGF-beta1, but not by anti-IL-10Rbeta mAb. Furthermore, IL-9 inhibited LPS-induced activation of extracellular signal-regulated kinase 1/2 mitogen-activated protein kinases in monocytes through a TGF-beta-mediated induction of protein phosphatase activity. In contrast, IL-4, which exerts a similar inhibitory effect on the oxidative burst and TNF-alpha release by monocytes, acts primarily through a down-regulation of LPS receptors. Thus, IL-9 deactivates LPS-stimulated blood mononuclear phagocytes, and the mechanism of inhibition involves the potentiation of TGF-beta1 production and extracellular signal-regulated kinase inhibition. These findings highlight a new target cell for IL-9 and may account for the beneficial activity of IL-9 in animal models of exaggerated inflammatory response.