A role for Bid in eosinophil apoptosis and in allergic airway reaction.

Bid, a proapoptotic member of Bcl-2 family, is involved in Fas receptor signaling. Fas activation promotes human eosinophil cell death and is believed to accelerate the resolution of pulmonary Th2-driven allergic reaction in mice. We hypothesized that Bid would regulate eosinophil apoptosis and Ag-induced airway inflammation, particularly eosinophilia. C57BL/6 Bid(-/-) and wild-type mice were immunized and repeatedly challenged with OVA, and bronchoalveolar lavage (BAL) fluid, lung, and spleen were collected 4-240 h after the final challenge. Cultured BAL eosinophils from Bid-deficient mice showed resistance to Fas-mediated apoptotic DNA fragmentation, phosphatidylserine exposure, mitochondria depolarization, and caspase-3 activity. In addition, OVA-challenged Bid(-/-) mice had higher BAL eosinophilia and a lower proportion of BAL apoptotic eosinophils than Bid(+/+) mice. This was accompanied by augmented BAL levels of the eosinophilotactic cytokine, IL-5, and of the eosinophil-associated mediators, TGF-beta1 and fibronectin. Finally, cultured OVA-stimulated lung mononuclear cells and splenocytes from Bid-deficient mice showed increased release of the Th2-type cytokines, IL-4 and IL-5, but no change in cell number. We conclude that Bid modulates BAL eosinophilia by regulating both eosinophil apoptosis and Th2-type cytokine production.

Defect of pro-hepatocyte growth factor activation by fibroblasts in idiopathic pulmonary fibrosis.

RATIONALE AND OBJECTIVES: Hepatocyte growth factor (HGF) protects against lung fibrosis in several animal models. Pro-HGF activation to HGF is subjected to regulation by its activator (HGFA), a serine protease, and HGFA-specific inhibitors (HAI-1 and HAI-2). Our hypothesis was that fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) had an altered capacity to activate pro-HGF in vitro compared with control fibroblasts. METHODS: We measured the kinetics of pro-HGF activation in human lung fibroblasts from control subjects and from patients with IPF by Western blot. HGFA, HAI-1, and HAI-2 expression was evaluated by immunohistochemistry, RNA protection assay, and Western blot. We evaluated the effect of TGF-beta(1) and PGE(2) on pro-HGF activation and HGFA, HAI-1, and HAI-2 expression. MAIN RESULTS: Lung fibroblasts activated pro-HGF in vitro. Pro-HGF activation was inhibited by serine protease inhibitors, by an anti-HGFA antibody, as well as by HAI-1 and HAI-2. Pro-HGF activation by IPF fibroblasts was reduced compared with control fibroblasts. In IPF fibroblasts, HGFA expression was lower and HAI-1 and HAI-2 expression was higher compared with control fibroblasts. PGE(2) stimulated pro-HGF activation through increased expression of HGFA and decreased expression of its inhibitor HAI-2. In contrast, TGF-beta(1) reduced the ability of lung fibroblasts to activate pro-HGF through decreased expression of HGFA and increased expression of its inhibitors. CONCLUSIONS: IPF fibroblasts have a low capacity to activate pro-HGF in vitro via a low level of HGFA expression and high levels of HAI-1 and HAI-2 expression, and PGE(2) is able to partially correct this defect.

Increased uptake of 111In-octreotide in idiopathic pulmonary fibrosis.

UNLABELLED: Idiopathic pulmonary fibrosis (IPF) is characterized by an uncontrolled accumulation and activation of lung fibroblasts. A modulation of fibroblast activation has been observed in various systems with octreotide, a synthetic somatostatin analog with strong affinity for the somatostatin receptor subtype 2 (sst2). One aim of our study was to evaluate the expression of somatostatin receptors in the lungs of patients with IPF. A second aim was to evaluate the relationship between 111In-octreotide uptake and the effect of pulmonary fibrosis as assessed by lung function tests and parameters and by radiologic findings. METHODS: We investigated 11 patients with IPF, 6 patients with pulmonary fibrosis associated with systemic sclerosis (SSc), and 19 patients with disease not of the lung (control patients). The expression of somatostatin receptors was evaluated in vivo using 111In-octreotide scintigraphy. We evaluated the relationship between 111In-octreotide uptake and the activity of pulmonary fibrosis as assessed by lung function tests, bronchoalveolar lavage (BAL) cellularity, and high-resolution CT (HRCT) of the chest. Planar images and thoracic SPECT (24 h) were performed after injection of 222 MBq of 111In-octreotide. Lung uptake was quantified using the lung-to-background ratio (L/B). In addition, the expression of sst2 was evaluated in vitro, in frozen lung-tissue samples using autoradiography, and in human cultures of lung fibroblasts using a ligand-binding assay. RESULTS: Compared with lung uptake in control patients (median L/B, 1.25; range, 1.14-1.49), lung uptake was increased in all 11 IPF patients (median L/B, 2.63; range, 1.59-3.13; P < 0.001) and in 4 of 6 SSc patients (median L/B, 1.68; range, 1.42-2.16). The L/B was lower in SSc patients than in IPF patients (P = 0.011). Increased uptake correlated with the alteration of lung function (carbon monoxide diffusing capacity [rho = -0.655; P = 0.038], diffusing capacity for carbon monoxide and alveolar volume ratio [rho = -0.627; P = 0.047], vital capacity [rho = -0.609; P = 0.054], and total lung capacity [rho = -0.598; P = 0.058]) and with the intensity of alveolitis (total BAL cellularity [rho = 0.756; P = 0.045], neutrophil counts [rho = 0.738; P = 0.05]), and HRCT fibrosis score (rho = 0.673; P = 0.007). Autoradiography suggested that vascular structures were a prominent binding site. Lung fibroblasts expressed somatostatin receptors in vitro as measured by binding assay. CONCLUSION: Our preliminary results identified an increased expression of sst2 in (mainly idiopathic) pulmonary fibrosis. Lung uptake correlates with the alteration of lung function and with the intensity of alveolitis.

Hepatocyte growth factor production by neutrophils infiltrating bronchioloalveolar subtype pulmonary adenocarcinoma: role in tumor progression and death.

Increased numbers of tumor-infiltrating neutrophils are linked to poorer outcome in patients with adenocarcinoma of the bronchioloalveolar carcinoma (BAC) subtype. Hepatocyte growth factor (HGF) is a pleiotropic cytokine operating through activation of the proto-oncogene c-met and is a factor of poor prognosis in various cancers. Reports that neutrophils produce HGF led us to investigate their participation in the aerogenous spread of tumor cells and the prognosis of BAC, through the effect of HGF on c-met-expressing tumor cells. Immunoreactive HGF was detected in bronchoalveolar lavage fluid (BALF) supernatants from 34 of 36 patients, whereas it was undetectable in BALF from healthy controls. The HGF thus detected was locally produced, because HGF mRNA was expressed by the patients' fresh alveolar cells, and HGF protein was detected in 24-h culture supernatants. In immunocytochemical studies of BALF cytospin preparations and tumor specimens from the patients, neutrophils were always HGF-positive, whereas alveolar macrophages and tumor cells gave inconsistent results. Alveolar neutrophil-derived HGFs induced significant, concentration-dependent migration of BAC-derived tumor cells in vitro, and this effect was inhibited by anti-HGF neutralizing antibodies. Granulocyte-macrophage colony-stimulating factor and tumor necrosis factor alpha (present in the lung tumor microenvironment) provoked HGF release from neutrophil intracellular stocks, and the capacity of blood neutrophils from BAC patients to produce HGF was unaltered. Immunochemical studies of c-met expression in BALF cytospin preparations and tumor sections showed that most HGF receptor-bearing cells were tumor cells. High HGF levels in BALF supernatants were significantly associated with poorer outcome in patients with BAC and were an independent predictor of clinical outcome in multivariate analysis. Altogether, our results support the notion that BAC generates a local environment that attracts functionally normal neutrophils from peripheral blood and leads to neutrophil release of biologically active HGF on contact with HGF receptor-expressing tumor cells, thereby contributing to poorer patient outcome.

Hepatocyte and keratinocyte growth factors and their receptors in human lung emphysema.

BACKGROUND: Hepatocyte and keratinocyte growth factors are key growth factors in the process of alveolar repair. We hypothesized that excessive alveolar destruction observed in lung emphysema involves impaired expression of hepatocyte and keratinocyte growth factors or their respective receptors, c-met and keratinocyte growth factor receptor. The aim of our study was to compare the expression of hepatocyte and keratinocyte growth factors and their receptors in lung samples from 3 groups of patients: emphysema; smokers without emphysema and non-smokers without emphysema. METHODS: Hepatocyte and keratinocyte growth factor proteins were analysed by immunoassay and western blot; mRNA expression was measured by real time quantitative polymerase chain reaction. RESULTS: Hepatocyte and keratinocyte growth factors, c-met and keratinocyte growth factor receptor mRNA levels were similar in emphysema and non-emphysema patients. Hepatocyte growth factor mRNA correlated negatively with FEV1 and the FEV1/FVC ratio both in emphysema patients and in smokers with or without emphysema. Hepatocyte and keratinocyte growth factor protein concentrations were similar in all patients' groups. CONCLUSION: The expression of hepatocyte and keratinocyte growth factors and their receptors is preserved in patients with lung emphysema as compared to patients without emphysema. Hepatocyte growth factor mRNA correlates with the severity of airflow obstruction in smokers.

Hepatocyte growth factor and lung fibrosis.

Idiopathic pulmonary fibrosis is currently believed to be driven by alveolar epithelial cells, with abnormally activated alveolar epithelial cells accumulating in an attempt to repair injured alveolar epithelium (1). Thus, targeting the alveolar epithelium to prevent or inhibit the development of pulmonary fibrosis might be an interesting therapeutic option in this disease. Hepatocyte growth factor (HGF) is a growth factor for epithelial and endothelial cells, which is secreted by different cell types, especially fibroblasts and neutrophils. HGF has mitogenic, motogenic, and morphogenic properties and exerts an antiapoptotic action on epithelial and endothelial cells. HGF has also proangiogenic effect. In vitro, HGF inhibits epithelial-to-mesenchymal cell transition and promotes myofibroblast apoptosis. In vivo, HGF has antifibrotic properties demonstrated in experimental models of lung, kidney, heart, skin, and liver fibrosis. Hence, the modulation of HGF may be an attractive target for the treatment of lung fibrosis.

Keratinocyte growth factor protects against elastase-induced pulmonary emphysema in mice.

Pulmonary emphysema is characterized by persistent inflammation and progressive alveolar destruction. The keratinocyte growth factor (KGF) favorably influences alveolar maintenance and repair and possesses anti-inflammatory properties. We aimed to determine whether exogenous KGF prevented or corrected elastase-induced pulmonary emphysema in vivo. Treatment with 5 mg x kg(-1) x day(-1) KGF before elastase instillation prevented pulmonary emphysema. This effect was associated with 1) a sharp reduction in bronchoalveolar lavage fluid total protein and inflammatory cell recruitment, 2) a reduction in the pulmonary expression of the chemokines CCL2 (or monocyte chemoattractant protein-1) and CXCL2 (or macrophage inflammatory protein-2alpha) and of the adhesion molecules ICAM-1 and VCAM-1, 3) a reduction in matrix metalloproteinase (MMP)-2 and MMP-9 activity at day 3, and 4) a major reduction in DNA damage detected by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) in alveolar cells at day 7. Treatment with KGF after elastase instillation had no effect on elastase-induced emphysema despite the conserved expression of the KGF receptor in the lungs of elastase-instilled animals as determined by immunohistochemistry. In vitro, KGF abolished the elastase-induced increase in CCL2, CXCL2, and ICAM-1 mRNA in the MLE-12 murine alveolar epithelial cell line. We conclude that KGF pretreatment protected against elastase-induced pulmonary inflammation, activation of MMPs, alveolar cell DNA damage, and subsequent emphysema in mice.

Keratinocyte growth factor expression by fibroblasts in pulmonary fibrosis: poor response to interleukin-1beta.

Keratinocyte growth factor (KGF) is secreted by fibroblasts and protects from pulmonary fibrosis in animal models. Interleukin (IL)-1beta is the most potent inducer of KGF in fibroblasts, acting through the c-Jun pathway. We evaluated in vitro KGF production by human lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF, n = 10) and from control subjects (n = 7) at baseline and after IL-1beta stimulation. Basal KGF secretion by IPF fibroblasts was similar to controls. In fibroblasts from control subjects, IL-1beta increased c-Jun expression, c-Jun activation, and KGF secretion. SP600125, a specific c-Jun N-terminal kinase (JNK) inhibitor, inhibited the effect of IL-1beta. By contrast, in IPF fibroblasts, IL-1beta did not increase c-Jun expression and c-Jun activation, and weakly increased KGF secretion, whereas SP600125 had no effect. IL-1beta similarly increased JunB expression in fibroblasts from patients with IPF and control subjects. Total JNK content was not different in either unstimulated or IL-1beta-stimulated IPF and control fibroblasts. IL-1beta increased phosphorylated JNK in control and IPF fibroblasts, but this increase was weaker and heterogeneous in IPF. Altogether, our results demonstrate a dysregulation of KGF secretion by IPF fibroblasts. The weak response to IL-1beta is associated with a defect of c-Jun expression and activation and a defect of JNK activation.

Three-dimensional characterization of pathologic lesions in pulmonary langerhans cell histiocytosis.

The characteristic lesions of pulmonary Langerhans cell histiocytosis (LCH) associate destructive granulomas containing large numbers of Langerhans cells and cysts. The lesions are usually considered to develop around small airways, and cysts are thought to result from destruction of the bronchiolar wall by the granulomatous reaction. However, the extent to which the granulomatous reaction is truly bronchocentric remains unknown, and the mode of formation of the cysts has not been defined. By using serial sections, this study aimed to explore further the relationships between pulmonary LCH lesions and distal airways, and the development of cysts. The results demonstrated that the granulomatous process of pulmonary LCH affected exclusively small airways, in an acinar distribution. The lesions extended without interruption along the bronchiolar axis, forming a continuous sheath around distal airways. The granulomatous reaction seemed to progress along the bronchiolar axis over time, extending the abnormalities in both the proximal and distal directions. Cystic lesions resulted from the destruction of the bronchiolar wall and progressive dilatation of the lumen, subsequently circumscribed by fibrous tissue. Because pulmonary LCH lesions affect and progressively destroy distal airways, it may be proper to consider the disease a bronchiolitis rather than an interstitial lung disorder.

Defect of hepatocyte growth factor secretion by fibroblasts in idiopathic pulmonary fibrosis.

Hepatocyte growth factor (HGF) is a growth factor that protects alveolar epithelial cells from pulmonary fibrosis in various animal models. We compared in vitro HGF production by human lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF, n = 8) and from control subjects (n = 6). Basal HGF secretion by IPF fibroblasts was decreased by 50% when compared with control fibroblasts (p < 0.05). HGF was secreted mainly in the cleaved mature form, both in IPF and control fibroblasts. HGF messenger RNA levels were reduced in IPF fibroblasts. Prostaglandin (PG) E2 secretion by IPF fibroblasts was low when compared with control subjects (p < 0.05). After the addition of PGE2 (10-6 M) or dibutyryl cyclic AMP (10-3 M), HGF secretion by IPF fibroblasts reached the level of control subjects. Inhibition of PGE2 synthesis with indomethacin reduced HGF secretion by control fibroblasts but had no effect on IPF fibroblasts. HGF secretion by control fibroblasts was also slightly inhibited by transforming growth factor (TGF)-beta1 and stimulated by anti-TGF-beta antibody, whereas both agents had no effect on IPF fibroblasts. Our results demonstrate a defect in HGF production by IPF fibroblasts that seems secondary to a defect in PGE2 secretion.

Differential role of neutrophils and alveolar macrophages in hepatocyte growth factor production in pulmonary fibrosis.

Neutrophils may participate in the development of lung fibrosis. Hepatocyte growth factor (HGF), a growth factor for type II pneumocytes, is produced by neutrophils. We measured the production of HGF by blood and alveolar neutrophils from patients with either idiopathic pulmonary fibrosis (n = 11) or connective tissue disease-associated pulmonary fibrosis (n = 10) and from control patients (n = 10). HGF secretion by alveolar macrophages and the expression of the HGF receptor by alveolar epithelial cells in pulmonary fibrosis were also evaluated. HGF was not detected in bronchoalveolar lavage fluid from controls. HGF concentration in the epithelial lining fluid from patients was 4-fold higher than in plasma, suggesting a local production within the alveolar space. Alveolar neutrophils secreted HGF in vitro. Basal HGF secretion by alveolar neutrophils positively correlated with HGF in the epithelial lining fluid (p = 0.05, rho = 0.582). HGF secretion by alveolar neutrophils could not be further stimulated with lipopolysaccharide, whereas HGF secretion by blood neutrophils doubled with lipopolysaccharide. Alveolar macrophages did not secrete HGF in vitro. The expression of the HGF receptor was greatly increased in the fibrotic lung, supporting the local function of HGF secreted by neutrophils. We conclude that neutrophils are a source of HGF in patients with pulmonary fibrosis.