Anna Maria Merendino

Evaluation of apoptosis of eosinophils, macrophages, and T lymphocytes in mucosal biopsy specimens of patients with asthma and chronic bronchitis

BACKGROUND Apoptosis regulates inflammatory cell survival, and its reduction contributes to the chronicity of an inflammatory process. Apoptosis is controlled by suppressing or inducing genes, such as bcl-2 and p53, respectively. OBJECTIVE We sought to assess apoptosis of eosinophils, macrophages, and T lymphocytes in bronchial biopsy specimens from asthmatic subjects and to examine its regulation by evaluating the expression of B-cell lymphoma leukemia-2 (Bcl-2) and P53 proteins. We also sought to explore the relationships between cell apoptosis and GM-CSF, a cytokine able to increase eosinophil and macrophage survival. METHODS Apoptosis in eosinophils, macrophages, and T lymphocytes was evaluated in bronchial biopsy specimens obtained from 30 asthmatic subjects, 26 subjects with chronic bronchitis, and 15 control subjects by combining the terminal deoxynucleotidyl transferase-mediated dNTP nick end-labeling technique and immunohistochemistry. The expression of P53, Bcl-2, and GM-CSF was studied through immunohistochemistry by using specific mAbs. RESULTS The number of apoptotic eosinophils and macrophages was lower in subjects with asthma than in those with chronic bronchitis (P <.007 and P <.001, respectively) and inversely correlated with the clinical severity of asthma (P <.001 and P <.002, respectively). Few T lymphocytes were apoptotic in all groups studied. In asthma GM-CSF+ cells correlated with the number of nonapoptotic eosinophils and macrophages (P =.0001) and with the severity of the disease (P <.003). In asthma Bcl-2+ cells were higher than in control subjects and subjects with chronic bronchitis (P <.002 and P <.015, respectively), they outnumbered P53+ cells, and they correlated with the number of T lymphocytes (P <.001) and with the severity of the disease (P <.003). CONCLUSION Airway inflammation in asthma is associated with an enhanced survival of different cell types caused by reduced apoptosis.

Hsp60 is actively secreted by human tumor cells

Background Hsp60, a Group I mitochondrial chaperonin, is classically considered an intracellular chaperone with residence in the mitochondria; nonetheless, in the last few years it has been found extracellularly as well as in the cell membrane. Important questions remain pertaining to extracellular Hsp60 such as how generalized is its occurrence outside cells, what are its extracellular functions and the translocation mechanisms that transport the chaperone outside of the cell. These questions are particularly relevant for cancer biology since it is believed that extracellular chaperones, like Hsp70, may play an active role in tumor growth and dissemination. Methodology/Principal Findings Since cancer cells may undergo necrosis and apoptosis, it could be possible that extracellular Hsps are chiefly the result of cell destruction but not the product of an active, physiological process. In this work, we studied three tumor cells lines and found that they all release Hsp60 into the culture media by an active mechanism independently of cell death. Biochemical analyses of one of the cell lines revealed that Hsp60 secretion was significantly reduced, by inhibitors of exosomes and lipid rafts. Conclusions/Significance Our data suggest that Hsp60 release is the result of an active secretion mechanism and, since extracellular release of the chaperone was demonstrated in all tumor cell lines investigated, our observations most likely reflect a general physiological phenomenon, occurring in many tumors.

Release of transforming growth factor‐beta (TGF‐β) and fibronectin by alveolar macrophages in airway diseases

Asthma and chronic bronchitis are associated with airway remodelling, and airway macrophages are present in bronchial inflammation. TGF‐β and fibronectin released by alveolar macrophages possess a fibrogenic potency. The potential role of alveolar macrophages in airway remodelling was studied in asthma and chronic bronchitis by the release of TGF‐β and fibronectin. Alveolar macrophages were isolated by bronchoalveolar lavage in 14 control subjects, 14 asthmatics and 14 chronic bronchitics. The spontaneous and lipopolysaccharide (LPS)‐ or concanavalin A (Con A)‐induced release of TGF‐β and fibronectin was measured by ELISA. Alveolar macrophages from chronic bronchitics spontaneously release greater amounts of TGF‐β and fibronectin than those from asthmatic and control subjects. Alveolar macrophages from asthmatics release greater amounts of TGF‐β and fibronectin than those from control subjects. The spontaneous release of TGF‐β is significantly correlated with that of fibronectin. Fibronectin release was significantly reduced after LPS stimulation, and TGF‐β release was significantly increased after LPS stimulation, except in chronic bronchitis patients. Con A increased the release of TGF‐β in cells from normal subjects. This study suggests that activated macrophages play a role in airway remodelling in chronic bronchitis and to a lesser extent in asthma.

Heat shock protein-27 protects human bronchial epithelial cells against oxidative stress–mediated apoptosis: possible implication in asthma

Abstract Inflammation of the human bronchial epithelium, as observed in asthmatics, is characterized by the selective death of the columnar epithelial cells, which desquamate from the basal cells. Tissue repair initiates from basal cells that resist inflammation. Here, we have evaluated the extent of apoptosis as well as the Hsp27 level of expression in epithelial cells from bronchial biopsy samples taken from normal and asthmatic subjects. Hsp27 is a chaperone whose expression protects against oxidative stress. We report that in asthmatic subjects the basal epithelium cells express a high level of Hsp27 but no apoptotic morphology. In contrast, apoptotic columnar cells are devoid of Hsp27 expression. Moreover, we observed a decreased resistance to hydrogen peroxide–induced apoptosis in human bronchial epithelial 16–HBE cells when they were genetically modified to express reduced levels of Hsp27.

The odyssey of Hsp60 from tumor cells to other destinations includes plasma membrane-associated stages and Golgi and exosomal protein-trafficking modalities.

Background In a previous work we showed for the first time that human tumor cells secrete Hsp60 via exosomes, which are considered immunologically active microvesicles involved in tumor progression. This finding raised questions concerning the route followed by Hsp60 to reach the exosomes, its location in them, and whether Hsp60 can be secreted also via other mechanisms, e.g., by the Golgi. We addressed these issues in the work presented here. Principal Findings We found that Hsp60 localizes in the tumor cell plasma membrane, is associated with lipid rafts, and ends up in the exosomal membrane. We also found evidence that Hsp60 localizes in the Golgi apparatus and its secretion is prevented by an inhibitor of this organelle. Conclusions/Significance We propose a multistage process for the translocation of Hsp60 from the inside to the outside of the cell that includes a combination of protein traffic pathways and, ultimately, presence of the chaperonin in the circulating blood. The new information presented should help in designing future strategies for research and for developing diagnostic-monitoring means useful in clinical oncology.

Apoptosis and airway inflammation in asthma

Asthma is a disease characterized by a chronic inflammation of the airways and by structural alterations of bron-chial tissues, often referred to as airway remodelling. The development of chronic airway inflammation in asthma depends upon the continuous recruitment of inflammatory cells from the bloodstream towards the bronchial mucosa and by their subsequent activation. It is however increasingly accepted that mechanisms involved in the regulation of the survival and apoptosis of inflammatory cells may play a central role in the persistent inflammatory process characterizing this disease. Increased cellular recruitment and activation, enhanced cell survival and cell:cell interactions are therefore the key steps in the development of chronic airway inflammation in asthma, and represent the major causes for tissue damge, repair and remodelling.

Effect of indomethacin on the kinetics of tumour necrosis factor alpha release and tumour necrosis factor alpha gene expression by human blood monocytes

In this investigation we have examined the effects of indomethacin, an inhibitor of the cyclooxygenase pathway of arachidonic acid, upon the kinetics of the release of tumour necrosis factor alpha (TNF) and of the expression of TNF gene by lipopolysaccharide (LPS)-stimulated human blood monocytes (BM). Following stimulation of BM with LPS, TNF was released within 2 h, reached peak values at 8 h and declined at subsequent time-points (24 and 48 h). Indomethacin (10(-5) M) slightly stimulated the production of TNF at 2, 4, and 8 h and prevented the decline of TNF observed at 24 and 48 h. This effect was related to the persistence of TNF synthesis, as demonstrated by kinetic evaluation of the expression of TNF gene performed by dot-blot analysis. The effects of indomethacin on TNF release and TNF gene expression were due to the inhibition of endogenous prostaglandin (PG)E2 production. In the absence of indomethacin, PGE2 release by the LPS-stimulated BM began concomitantly with the decline of TNF production by the same cells under the same stimulus. Indomethacin completely blocked PGE2 release at any time-point. Exogenous PGE2 suppressed the release of TNF and the expression of TNF gene in a dose-dependent fashion. Exogenous PGE2 completely reversed the effects of indomethacin on TNF production at 24 h. These findings suggest that indomethacin may significantly alter the kinetics of TNF release and TNF gene expression by LPS-stimulated BM. These effects are related, at least in part, to the inhibition of the production of endogenous PGE2, an important self-driven regulatory factor of the kinetics of TNF production.

ICAM‐1 and α3β1 expression by bronchial epithelial cells and their in vitro modulation by inflammatory and anti‐inflammatory mediators

Background: Adhesion molecules are involved in inflammatory and repair processes of the bronchial epithelium. ICAM‐1 is mainly involved in inflammatory reactions, whereas integrins, such as α3β1, are mainly involved in repair processes.

IκB kinase–driven nuclear factor-κB activation in patients with asthma and chronic obstructive pulmonary disease

BACKGROUND Nuclear factor-κB (NF-κB) is a transcriptional factor of different inflammatory patterns involved in asthma and chronic obstructive pulmonary disease (COPD) that is tightly controlled by IκB kinase (IKK) complex. OBJECTIVE We investigated the dysregulation of IKK-driven NF-κB activation in patients with asthma and COPD. METHODS We assessed IKKα and IKKβ expression and activation, their regulation by glucocorticosteroids, and their involvement in IL-8 synthesis in PBMCs isolated from asthmatic patients, healthy smokers (HSs), patients with COPD, and control subjects. PBMCs from control subjects were stimulated with TNF-α and cigarette smoke extract in the presence or absence of fluticasone propionate (FP), L-glutathione reduced, or both, and IKK activation and IL-8 release were evaluated. RESULTS IKKα activity was higher in patients with COPD and HSs than in asthmatic patients and control subjects. IKKβ activity was higher in asthmatic patients, HSs, and patients with COPD than in control subjects. In vitro FP treatment induced inhibition of both IKKα and IKKβ activity in PBMCs from asthmatic patients, patients with COPD, and HSs, although IKKβ activity was more sensitive to FP than that of IKKα. FP reduced the IL-8 released from PBMCs of asthmatic patients, patients with COPD, and HSs, although IL-8 inhibition was higher in asthmatic patients than in patients with COPD and HSs. FP reduced IKKα and IKKβ activities in TNF-α and cigarette smoke extract-treated PBMCs, with higher levels of inhibition for IKKβ than IKKα activity. L-glutathione reduced improved the downregulatory effects of FP on IKKα and IL-8 levels. CONCLUSION Based on differential activation of IKKα and IKKβ, our findings suggest a different profile in the upstream regulation of the IKK-driven NF-κB system in asthmatic patients and patients with COPD. These differences in the regulation of the inflammatory process may explain, at least in part, the different pharmacologic responses in these patients.

Tumor Necrosis Factor Release by Pulmonary Alveolar Macrophages

Tumor necrosis factor (TNF) is a monokine with cytotoxic and tumor-necrosing activities that may play a role in host defence against malignancies. The aim of the present study was to assess the ability of pulmonary alveolar macrophages (AMs) to release TNF. To accomplish this, a cytotoxicity assay specific for TNF detection was applied, using as targets actinomycin-D-pretreated WEHI 164 murine sarcoma cells. AMs mediated cytotoxicity levels comparable to those mediated by autologous blood monocytes (BMs). In addition, cytotoxicity was accounted for by a soluble factor released by effector cells and was completely inhibited by the addition of anti-TNF antibodies. These results indicate that AMs are able to release TNF. Moreover, since TNF has now been recognized as an important mediator of inflammation, its production by AMs suggests that this monokine might play a role in the pathogenesis of a variety of nonmalignant lung disorders.