N.L.A. Misso

Clinical effects of sulphite additives

Sulphites are widely used as preservative and antioxidant additives in the food and pharmaceutical industries. Topical, oral or parenteral exposure to sulphites has been reported to induce a range of adverse clinical effects in sensitive individuals, ranging from dermatitis, urticaria, flushing, hypotension, abdominal pain and diarrhoea to life‐threatening anaphylactic and asthmatic reactions. Exposure to the sulphites arises mainly from the consumption of foods and drinks that contain these additives; however, exposure may also occur through the use of pharmaceutical products, as well as in occupational settings. While contact sensitivity to sulphite additives in topical medications is increasingly being recognized, skin reactions also occur after ingestion of or parenteral exposure to sulphites. Most studies report a 3–10% prevalence of sulphite sensitivity among asthmatic subjects following ingestion of these additives. However, the severity of these reactions varies, and steroid‐dependent asthmatics, those with marked airway hyperresponsiveness, and children with chronic asthma, appear to be at greater risk. In addition to episodic and acute symptoms, sulphites may also contribute to chronic skin and respiratory symptoms. To date, the mechanisms underlying sulphite sensitivity remain unclear, although a number of potential mechanisms have been proposed. Physicians should be aware of the range of clinical manifestations of sulphite sensitivity, as well as the potential sources of exposure. Minor modifications to diet or behaviour lead to excellent clinical outcomes for sulphite‐sensitive individuals.

Fibroblasts Isolated from Normal Lungs and Those with Idiopathic Pulmonary Fibrosis Differ in Interleukin-6/gp130-Mediated Cell Signaling and Proliferation

Interleukin (IL)-6 and IL-11 are elevated in a variety of lung conditions and may impact on repair mechanisms in chronic inflammatory disorders. However, the mechanisms by which these cytokines influence fibroblast proliferation in normal and disease states have not been previously addressed. We examined the effect of these cytokines on proliferation and cell-cycle kinetics of primary human lung fibroblasts obtained from normal patients and patients with idiopathic pulmonary fibrosis (IPF). IL-6 inhibited the proliferation of normal fibroblasts due to the sustained phosphorylation of STAT-3 and production of the cyclin-dependent kinase inhibitor p19(INK4D). In contrast IL-6 was mitogenic for IPF fibroblasts due to the sustained activation of MAPK, which in turn inhibited the production of p27(Kip1), allowing activation of cyclin D(1) and hyperphosphorylation of retinoblastoma protein. IL-11 was mitogenic for both normal and IPF fibroblasts. These results provide strong evidence for a fundamental abnormality in a cytokine-signaling pathway, as opposed to alterations in cytokine production, in the pathogenesis of IPF.

Macrophage recognition and phagocytosis of apoptotic fibroblasts is critically dependent on fibroblast-derived thrombospondin 1 and CD36

The induction of fibroblast apoptosis and their clearance by phagocytes is essential for normal wound healing and prevention of scarring. However, little is known about the clearance of apoptotic fibroblasts and whether apoptotic cells are active participants in the recruitment and activation of phagocytes. In this study, we provide the first evidence that apoptotic fibroblasts actively release increased amounts of thrombospondin (TSP1) to actively recruit macrophages. Expression of TSP1 and its receptor CD36 was increased on the surface of apoptotic fibroblasts. By chemical cross-linking and immunoprecipitation we show that TSP1 and CD36 were directly associated. This was confirmed by confocal microscopy. Blockade of either CD36 or TSP1 on apoptotic fibroblasts inhibited phagocytosis. Blockade of alpha v beta 3 integrins as well as CD36 and TSP1 on macrophages inhibited phagocytosis. In contrast, phosphatidylserine or lectins were not involved. These findings suggest that apoptotic fibroblasts release TSP1 as a signal to recruit macrophages while the up-regulated expression of the CD36/TSP1 complex on their cell surface may form a ligand bridging the fibroblast to a complex consisting of alpha v beta 3/CD36/TSP1 on macrophages. These results establish fundamental mechanisms for the clearance of apoptotic fibroblasts and may provide insights into the processes involved in normal wound repair.

Comparison of the morphological and biochemical changes in normal human lung fibroblasts and fibroblasts derived from lungs of patients with idiopathic pulmonary fibrosis during FasL-induced apoptosis.

It is increasingly recognized that the morphological changes of apoptosis vary between cell types. This heterogeneity reflects the wide range of cellular proteins and enzymes involved in apoptotic pathways. Fibroblast apoptosis is crucial to the regression of scars and the restitution of healthy tissue during wound repair and may be aberrant in diseases such as idiopathic pulmonary fibrosis (IPF). The biochemical and morphological changes characterizing fibroblast apoptosis are unknown and may provide insights into the specific enzymatic mediators activated in these cells. This study aimed to examine the morphological changes of fibroblast apoptosis in both primary normal lung fibroblasts (normal‐Fb) and fibroblasts obtained from patients with idiopathic pulmonary fibrosis (IPF‐Fb) and to correlate these changes with conventional biochemical markers. Transmission electron microscopy (TEM) and video time‐lapse microscopy demonstrated no difference in the duration of fibroblast apoptosis in response to FasL (6 ± 0.3 h in normal‐Fb and 6.4 ± 0.2 h in IPF‐Fb). However, IPF‐Fb were more resistant to FasL‐induced apoptosis compared with normal‐Fb. Although the majority of morphological changes of normal‐Fb and IPF‐Fb were similar, the formation of filopodia and condensation of the cytoskeletal bundles in IPF‐Fb, and more prominent vacuolation in normal‐Fb, were the significant differences between these cell subtypes. Loss of the mitochondrial membrane potential occurred prior to caspase‐3 activation, while phosphatidylserine expression, cytokeratin‐18 cleavage, and DNA fragmentation commenced after caspase‐3 activation. These observations not only suggest that specific enzymatic effectors may be preferentially activated during fibroblast apoptosis, but also provide potential insights into the pathogenesis of IPF. Copyright

PGE2 and dibutyryl cyclic adenosine monophosphate prolong eosinophil survival in vitro

BACKGROUND Apoptosis represents a mechanism by which the accumulation and inflammatory potential of eosinophils in asthma might be limited. Mediators derived from the airway epithelium may influence the rate of eosinophil apoptosis. OBJECTIVE We have investigated the effects on eosinophil apoptosis of 3 mediators that are likely to be produced by the airway epithelium, namely PGE2, TNF-alpha, and nitric oxide. METHODS Peripheral blood eosinophils from healthy adult volunteers were purified by density gradient centrifugation and negative immunomagnetic selection. Eosinophils were cultured for 16 or 40 hours with PGE2 (10 nmol/L), dibutyryl cyclic adenosine monophosphate (AMP; 100 micromol/L), TNF-alpha (500 U/mL), the nitric oxide donors, S-nitroso-N-acetylpenicillamine (100 micromol/L), and 2,2;-(hydroxynitrosohydrazono)bis-ethanamine (1 mmol/L), or dibutyryl cyclic guanosine monophosphate (100 micromol/L). Control cultures consisted of untreated, IL5-treated (100 U/mL), and anti-Fas-treated (400 ng/mL) cells. Eosinophil apoptosis was assessed by flow cytometric analysis of annexin V-FITC binding to externalized phosphatidylserine, by electrophoresis of phosphorus 32 end-labeled DNA fragments, and by flow cytometric assessment of hypodiploid DNA with propidium iodide. RESULTS PGE2 and cyclic AMP inhibited spontaneous eosinophil apoptosis at both 16 and 40 hours as did the PGEP2 receptor agonist, 11-deoxy PGE1, at 40 hours, but these effects were not inhibited by a protein kinase A antagonist. TNF-alpha delayed apoptosis in eosinophil cultures at 16 hours, whereas S-nitroso- N-acetylpenicillamine, 2, 2;-(hydroxynitrosohydrazono)bis-ethanamine, and cyclic guanosine monophosphate had little effect. Anti-Fas had little effect on spontaneous eosinophil apoptosis but significantly reduced the inhibitory effects of PGE2, cyclic AMP, and TNF-alpha. Assessments of apoptosis by DNA fragmentation gave similar but quantitatively less sensitive results. CONCLUSION Inhibition of spontaneous eosinophil apoptosis by PGE2 appears to be mediated by EP2 receptors but is not protein kinase A dependent. By enhancing eosinophil survival, PGE2 may increase the proinflammatory potential of these cells in chronic asthma.

Expression of tissue and plasma kallikreins and kinin B1 and B2 receptors in lung cancer

Abstract Tissue kallikrein (hK1) and plasma kallikrein (PK, hKB1) are serine proteases that produce biologically active kinin peptides from endogenous kininogen substrates. There is evidence linking the kallikreins and the mitogenic kinin peptides to carcinogenesis. The aim of this study was to investigate the expression of tissue prokallikrein (pro-hK1), plasma prekallikrein (PPK, pre-hKB1) and kinin B1 and B2 receptor proteins in different subtypes of lung cancer. Immunohistochemistry, using specific antibodies, was performed on archived normal lung sections and sections from adenocarcinomas, squamous cell carcinomas, large cell carcinomas, small cell carcinomas and carcinoid tumours of the lung. Immunoperoxidase labelling was visualised by brightfield microscopy and immunofluorescence labelling by confocal microscopy. Extensive cytoplasmic expression of pro-hK1 and PPK was observed, which was similar in small cell and non-small cell tumours. However, nuclear labelling for the kallikreins was absent or limited. The kinin B1 and B2 receptors were highly expressed in the cytoplasm of all tumour types and in the nuclei of non-small cell tumours. Further studies are required to assess the functional significance of the expression of hK1, PK and kinin receptors in lung tumours, and whether any of these proteins may be potential biomarkers for specific subtypes of lung carcinoma.

Prostaglandin E2 and cysteinyl leukotriene concentrations in sputum: association with asthma severity and eosinophilic inflammation

Background Inflammation of the airways in asthma is associated with the production of cysteinyl leukotrienes (cysLT), prostaglandin (PG)E2, 8‐isoprostane, nitric oxide and other mediators. However, the relationship between asthma severity or eosinophilic inflammation and the concentrations of mediators in sputum is unclear.

Oxidant stress induces gamma‐glutamylcysteine synthetase and glutathione synthesis in human bronchial epithelial NCI‐H292 cells

Background The bronchial epithelium is exposed to reactive oxygen species (ROS) derived from cigarette smoke, air pollutants and activated leucocytes. Glutathione (GSH) prevents ROS‐mediated loss of cell function, tissue injury and inflammation, and its synthesis is regulated by γ‐glutamylcysteine synthetase (γ‐GCS). However, the capacity of bronchial epithelial cells to adapt to oxidative stress and the mechanisms involved are not known.

The effect of eicosapentaenoic acid consumption on human neutrophil chemiluminescence

The effect of eicosapentaenoic acid (EPA) on the inflammatory potential of neutrophils was investigated by supplementing the diets of 12 subjects with 2.16 g of EPA or 12 g of olive oil per day for 4 weeks in a double blind crossover study. Neutrophil function as assessed by luminol enhanced chemiluminescence responses to plateletactivating factor (PAF) and formyl-methionyl-leucyl-EPA but not after olive oil consumption in the subjects who consumed EPA first. In contrast, EPA had no significant effect on neutrophil chemiluminescence in the subjects who consumed olive oil first. Dietary supplementation with EPA inhibits neutrophil responses to inflammatory mediators such as PAF while other fatty acids appear to modify the effects of EPA.

Differential expression of neutrophil adhesion molecules during coronary artery surgery with cardiopulmonary bypass

BACKGROUND Activation of neutrophil adhesion molecules and subsequent neutrophil adhesion to vascular endothelium are key events initiating inflammatory organ dysfunction after cardiopulmonary bypass and ischemic reperfusion. OBJECTIVES We sought to characterize neutrophil integrin CD11b and L-selectin activation associated with coronary artery bypass graft surgery and to determine whether neutrophil activation contributes to their sequestration on postbypass reperfusion. METHODS Twenty patients undergoing routine coronary artery bypass were studied. Heparinized whole blood was simultaneously sampled from a central venous line, aorta, coronary sinus, and right and left atrium before, during, and up to 20 minutes after cardiopulmonary bypass. Neutrophil counts were obtained, and neutrophil CD11b and L-selectin expression was determined by flow cytometric analysis in whole blood. RESULTS CD11b expression on circulating neutrophils increased during cardiopulmonary bypass, peaking at 145% of baseline level after release of the aortic clamp and then declined by 20 minutes after bypass (analysis of variance, P =.003). No change in neutrophil L-selectin expression was observed during cardiopulmonary bypass. Neutrophils responded to ex vivo stimulation by C5a and leukotriene B(4) during cardiopulmonary bypass but not at 24 hours after the operation. After reperfusion, neutrophil loss, but not local activation, was demonstrated in the coronary and pulmonary circulations. CONCLUSIONS Upregulated CD11b expression on neutrophils is likely to contribute to neutrophil sequestration in the heart and lungs after bypass, but neutrophil activation may be limited by their reduced responsiveness to agonist stimulation. CD11b represents a potential therapeutic target for diminishing inflammation after cardiac operations.