Jun-ichi Ashitani

Increased concentrations of human beta-defensins in plasma and bronchoalveolar lavage fluid of patients with diffuse panbronchiolitis.

Background: Human β-defensin (HBD)-1 and -2 are antimicrobial peptides present in the respiratory tract. Recent reports have indicated reduced activity of β-defensins in cystic fibrosis, suggesting that β-defensins may play an important role in the pathological process of chronic respiratory tract infection. Diffuse panbronchiolitis (DPB) is a progressive disease characterised by frequent episodes of superimposed infection, typically caused by Pseudomonas aeruginosa. The aim of this study was to elucidate the role of these antimicrobial peptides in this disease. Methods: The concentrations of HBD-1 and HBD-2 in plasma and bronchoalveolar lavage (BAL) fluid from 33 patients with DPB and 30 normal adults were measured by radioimmunoassay. Localisation of HBD-2 was investigated immunohistochemically in an open lung biopsy specimen obtained from a patient with DPB. Results: High concentrations of HBD-1 and HBD-2 were noted in BAL fluid from DPB patients. Increased plasma concentrations of HBD-2, but not HBD-1, were found in patients with DPB compared with control subjects. In patients with DPB the HBD-2 concentration in BAL fluid correlated significantly with the numbers of cells recovered from the BAL fluid (total cells, neutrophils, and lymphocytes) and with the BAL fluid concentration of IL-1β. Synthetic HBD-2, but not HBD-1, had dose dependent bactericidal activity against P aeruginosa. Treatment of 14 patients with macrolides significantly reduced BAL fluid concentrations of HBD-2 but not HBD-1 or plasma concentrations of HBD-1 and HBD-2. Immunohistochemistry of lung tissue showed localisation of HBD-2 in the epithelia of the distal bronchioles. Conclusions: These results indicate that β-defensins, particularly HBD-2, participate in antimicrobial defence in the respiratory tract in DPB, and that the BAL fluid concentration of HBD-2 may be a useful marker of airway inflammation in patients with DPB.

Elevated concentrations of defensins in bronchoalveolar lavage fluid in diffuse panbronchiolitis

Human neutrophils contain three isoforms of antimicrobial and cytotoxic peptides in the azurophil granules, which belong to a family of mammalian neutrophil peptides named defensins. Here we investigate the role of these peptides in diffuse panbronchiolitis (DPB). Defensins (human neutrophil peptide-1, -2 and -3) were measured by radioimmunoassay in bronchoalveolar lavage fluid (BALF) of 30 patients with DPB, 16 patients with idiopathic pulmonary fibrosis (IPF) and 15 healthy adults. The concentration of defensins was higher in BALF of patients with DPB than in patients with IPF and healthy subjects. DPB and IPF patients also had significantly higher plasma concentrations of defensins than controls. In patients with DPB, BALF concentration of defensins correlated significantly with neutrophil count or BALF concentration of interleukin (IL)-8. Immunohistochemistry of open-lung biopsy specimens from four DPB patients showed localization of defensins in neutrophils and mucinous exudate in the airways, and on the surface of bronchiolar epithelial cells. In vitro studies showed an enhanced extracellular release of defensins following stimulation of neutrophils with phorbol myristate acetate, N-formyl-methionyl-leucyl-phenyalamine, and human recombinant IL-8. Treatment of DPB with macrolides for 6 months significantly reduced neutrophil count and concentrations of defensins and IL-8 in BALF. Our results indicate accumulation of neutrophil-derived defensins in the airway in diffuse panbronchiolitis, and suggest that defensins may be a marker of neutrophil activity in this disease.

Raised plasma concentrations of α-defensins in patients with idiopathic pulmonary fibrosis

Background: Neutrophils are thought to play an important role in the pathogenesis of idiopathic pulmonary fibrosis (IPF). Human neutrophils contain antimicrobial and cytotoxic peptides in the azurophil granules which belong to a family of mammalian neutrophil peptides named α-defensins. A study was undertaken to investigate the role of α-defensins in the pathogenesis of IPF. Methods: The concentrations of α-defensins (human neutrophil peptides (HNPs) 1, 2, and 3) in plasma and bronchoalveolar lavage (BAL) fluid of 30 patients with IPF and 15 healthy subjects were measured by radioimmunoassay. Results: The concentrations of α-defensins in plasma, but not in BAL fluid, were significantly higher in IPF patients than in controls. BAL fluid concentrations of interleukin (IL)-8 in patients with IPF, which were significantly higher than in controls, correlated with those of α-defensins. An inverse relationship was seen between plasma α-defensin levels and the arterial oxygen tension (Pao2) and pulmonary function (vital capacity (%VC), forced expiratory volume in 1 second (FEV1), and carbon monoxide transfer factor (%Tlco)) in patients with IPF. Plasma levels of α-defensins also correlated with the clinical course in IPF patients with an acute exacerbation. Immunohistochemically, positive staining was observed inside and outside neutrophils in the alveolar septa, especially in dense fibrotic areas. Conclusion: These findings suggest that α-defensins play an important role in the pathogenesis of IPF, and that the plasma α-defensin level may be a useful marker of disease severity and activity.

Ghrelin treatment suppresses neutrophil-dominant inflammation in airways of patients with chronic respiratory infection.

BACKGROUND Persistent neutrophil influx into the airways is a characteristic of chronic respiratory infection and contributes to the deterioration of pulmonary function. Ghrelin is a novel growth hormone (GH)-releasing peptide with potential anti-inflammatory activities. The present study investigated whether or not ghrelin can reduce neutrophil-dominant inflammation in airways of patients with chronic respiratory infection. POPULATIONS AND METHODS Synthesized ghrelin was administered intravenously for 3 weeks to 7 cachectic patients with chronic respiratory infection to confirm ghrelins effects on airway inflammation and nutrition state. Neutrophils, neutrophil products and inflammatory cytokines in sputum were used as markers of airway inflammation. Changes in serum protein levels were also evaluated along with plasma catecholamine levels. Exercise tolerance was assessed by measuring 6-min walking distance before and after 3 weeks of ghrelin treatment. RESULTS Three-week ghrelin administration decreased neutrophil density and inflammatory cytokine levels in sputum, reduced plasma norepinephrine level, and increased body weight, serum protein level, and 6-min walking distance. CONCLUSIONS Ghrelin administration suppressed airway inflammation by decreasing neutrophil accumulation in lungs and increased body weight. These findings may contribute to the development of supportive therapies for patients with refractory chronic respiratory infection.

Identification of hBD-3 in respiratory tract and serum: the increase in pneumonia

Human β-defensin (hBD)-3, a 45 amino acid antimicrobial peptide, was originally isolated from human skin. hBD-3 mRNA has also been detected in the airways by RT-PCR. While hBD-3 may be involved in antimicrobial defences within the respiratory tract, the presence of hBD-3 peptide in the respiratory system has not yet been confirmed. The antimicrobial activity of the synthesised hBD-3 peptide was measured by a radial diffusion assay and a colony count assay. The present authors confirmed the presence of hBD-3 peptide in homogenates of human lung and serum using reverse-phase HPLC coupled with a highly sensitive RIA. The localisation of the hBD-3 peptide was investigated by immunohistochemistry. In addition, the serum concentrations of hBD-3 were measured by RIA. hBD-3 exhibited a strong antimicrobial activity, which was unaffected by increasing salt concentrations. Immunohistochemically, the current authors observed the expression of hBD-3 in bronchial and bronchiolar epithelial cells. The mean±sd serum concentration of hBD-3 in patients with bacterial pneumonia was 239.4±17.8 pg·mL−1 in the acute phase and, decreased to 159.3±20.1 pg·mL−1 after the completion of therapy. In conclusion, these findings will help elucidate the role of human β-defensin-3 in host immune responses and identify the pathophysiological significance of this molecule in respiratory infections.

Ghrelin ameliorates bleomycin-induced acute lung injury by protecting alveolar epithelial cells and suppressing lung inflammation

Acute lung injury is a critical illness syndrome consisting of acute respiratory failure with bilateral pulmonary infiltrates that is refractory to current therapies. Acute lung injury is characterized by injury of the alveolar capillary barrier, neutrophil accumulation, and induction of pro-inflammatory cytokines followed by devastating lung fibrosis. Ghrelin, an acylated peptide produced in the stomach, increases food intake and growth hormone secretion, suppresses inflammation, and promotes cell survival. We investigated the pharmacological potential of ghrelin in the treatment of acute lung injury by using a bleomycin-induced acute lung injury model in mice. Ghrelin or saline was given to mice daily starting 1 day after bleomycin administration. Ghrelin-treated mice showed a definitively higher survival rate than saline-treated ones. They also had smaller reductions in body weight and food intake. The amelioration of neutrophil alveolar infiltration, pulmonary vascular permeability, induction of pro-inflammatory cytokines, and subsequent lung fibrosis were notable in ghrelin-treated mice. Additionally, ghrelin administration reduced the injury-induced apoptosis of alveolar epithelial cells. Our results indicate that ghrelin administration exerts a protective effect against acute lung injury by protecting the alveolar epithelial cells and regulating lung inflammation, and highlight ghrelin as a promising therapeutic agent for the management of this intractable disease.

Ghrelin and its therapeutic potential for cachectic patients.

The discovery of ghrelin has resulted in the development of approaches to appetite, enabling a better understanding of the mechanisms regulating appetite through molecular analyses. Ghrelin is a 28-amino acid peptide that was isolated from the stomach only a decade ago, and has recently been investigated as a potential therapeutic endogenous agent. This peptide increases appetite, adjusts energy balance, suppresses inflammation, and enhances the release of growth hormone from the pituitary gland. Although many bioactive substances such as peptide YY, leptin, adiponectin and obestatin are involved in appetite control, ghrelin is the only known peptide to signal starvation information from a peripheral organ to the central nervous system, contributing to an increase in appetite. Clinical trials have revealed the effectiveness of ghrelin in increasing lean body mass and activity in cachectic patients. As shown in clinical research on humans and basic research using animal models, cachexia often occurs in response to excess release of proinflammatory cytokines and induces further appetite loss, which aggravates the physiological status of underlying diseases. Ghrelin functions as a protector against the vicious cycle of the cachectic paradigm through orexigenic, anabolic and anti-inflammatory effects, so administration of ghrelin may be able to improve quality of life in cachectic patients. We show here a significant role of ghrelin in the pathophysiology of cachectic diseases and the possibility of clinical applications.

Isolation of human β-defensin-4 in lung tissue and its increase in lower respiratory tract infection

BackgroundHuman β-defensin-4 (hBD-4), a new member of the β-defensin family, was discovered by an analysis of the genomic sequence. The objective of this study was to clarify hBD-4 expression in human lung tissue, along with the inducible expression in response to infectious stimuli, localization, and antimicrobial activities of hBD-4 peptides. We also investigated the participation of hBD-4 in chronic lower respiratory tract infections (LRTI) by measuring the concentrations of hBD-4 peptides in human bronchial epithelial lining fluid (ELF).MethodsThe antimicrobial activity of synthetic hBD-4 peptides against E. coli and P. aeruginosa was measured by radial diffusion and colony count assays. We identified hBD-4 in homogenated human lung tissue by reverse-phase high-performance liquid chromatography coupled with a radioimmunoassay (RIA). Localization of hBD-4 was studied through immunohistochemical analysis (IHC). We investigated the effects of lipopolysaccharide (LPS) on hBD-4 expression and its release from small airway epithelial cells (SAEC). We collected ELF from patients with chronic LRTI using bronchoscopic microsampling to measure hBD-4 concentrations by RIA.ResultshBD-4 exhibited salt-sensitive antimicrobial activity against P. aeruginosa. We detected the presence of hBD-4 peptides in human lung tissue. IHC demonstrated the localization of hBD-4-producing cells in bronchial and bronchiolar epithelium. The levels of hBD-4 peptides released from LPS-treated SAECs were higher than those of untreated control cells. ELF hBD-4 was detectable in 4 of 6 patients with chronic LRTI, while the amounts in controls were all below the detectable level.ConclusionThis study suggested that hBD-4 plays a significant role in the innate immunity of the lower respiratory tract.

Elevated levels of circulating adhesion molecules in patients with active pulmonary tuberculosis

Objective:  Recent studies have indicated the importance of cell adhesion molecules in the pathogenesis of various inflammatory lung diseases. Our study was designed to determine whether five soluble adhesion molecules including soluble L‐, E‐ and P‐selectin (sL‐, sE‐ and sP‐selectin), intercellular adhesion molecule‐1 (sICAM‐1), and vascular cell adhesion molecule‐1 (sVCAM‐1) in serum reflect the severity of active pulmonary tuberculosis (TB), and whether there is a distinct profile of these soluble molecules in this disease.

Increased Levels of Interleukin-18 in Bronchoalveolar Lavage Fluid of Patients with Idiopathic Nonspecific Interstitial Pneumonia

Background: Interleukin-18 (IL-18) is a proinflammatory cytokine that can induce interferon-γ (IFN-γ), and it plays an important role in T-helper 1 responses. Among idiopathic interstitial pneumonia (IIP), nonspecific interstitial pneumonia (NSIP) has an increased number of lymphocytes in bronchoalveolar lavage (BAL) fluid compared with usual interstitial pneumonia (UIP). However, the difference in their pathogenesis is unclear. Objective: The present study aims to investigate the roles of IL-18 in patients with idiopathic UIP and idiopathic NSIP. Methods: We measured the serum and BAL fluid (BALF) levels of IL-18 and IFN-γ in 22 patients with IIP (12 with UIP, 10 with NSIP) and 9 healthy volunteers. Results: Lymphocyte proportions in BALF were significantly higher in NSIP than in UIP and healthy subjects. No significant differences were observed in the serum IL-18 levels of all subjects, while the BALF levels of IL-18 in patients with NSIP were significantly higher than in patients with UIP (p < 0.005) and in healthy subjects (p < 0.005). Among all subjects, the levels of IL-18 in BALF correlated significantly with those in serum and the lymphocyte proportions in BALF. The serum IFN-γ levels of all subjects were below sensitivity, but there was significant reverse correlation between the levels of IFN-γ and the lymphocyte proportions in BALF. Conclusion: The lymphocytosis in BALF of patients with idiopathic NSIP and a part of idiopathic UIP might be associated with the high levels of IL-18.