Ryo Shirai

Interleukin 1β, Tumor Necrosis Factor Alpha, and Interleukin 8 in Bronchoalveolar Lavage Fluid of Patients with Diffuse Panbronchiolitis: A Potential Mechanism of Macrolide Therapy

We measured the levels of interleukin (IL) 1 beta, tumor necrosis factor alpha, and IL-8 in bronchoalveolar lavage fluid (BALF) and sera of patients with diffuse panbronchiolitis (DPB) before and after administration of erythromycin or roxithromycin. The pretreatment levels of IL-1 beta and IL-8 were significantly higher in the BALF of patients with DPB than in the BALF of patients with sarcoidosis and controls. The tumor necrosis factor alpha level was also higher than in controls, but not statistically significant. There was a significant correlation between percentage of neutrophils and IL-8 level in the BALF of DPB patients (r = 0.509; p < 0.05) on the one hand and between IL-1 beta and IL-8 on the other (r = 0.476; p < 0.04). Treatment for 1-24 months significantly reduced BALF levels of IL-1 beta and IL-8 of DPB patients in parallel with a reduction in BALF neutrophils. The serum level of IL-8 of DPB patients was higher, albeit insignificant, than that of controls and significantly lower than that in the BALF of the same patients (p = 0.0088). Serum IL-1 beta was below the detection limit. In addition, the concentration of IL-8 in alveolar macrophages obtained from 2 volunteers before and after oral erythromycin administration also decreased ex vivo. Our results indicate that IL-8 induces the migration of neutrophils to inflammatory sites. It is possible that the macrolides impair production and/or secretion of these cytokines, ultimately reducing neutrophil accumulation in the airway.

Differing effects of clarithromycin and azithromycin on cytokine production by murine dendritic cells

The macrolide antibiotics are now well known to have anti‐inflammatory effects. Because dendritic cells (DCs) orchestrate immune responses, we examined the in vitro effects of clarithromycin (CAM), azithromycin (AZM) and midecamycin (MDM) on the expression of co‐stimulatory molecules and production of cytokines [interleukin (IL)‐10, IL‐6, interferon (IFN)‐γ, IL‐12p40, tumour necrosis factor (TNF)‐α] of murine bone marrow‐derived DCs by lipopolysaccharide (LPS) stimulation. A 15‐membered macrolide, AZM, and a 14‐membered macrolide, CAM, significantly enhanced the intensity of a co‐stimulatory molecule, CD80, on DCs but not CD86 and CD40. AZM significantly increased the production of IL‐10 and CAM significantly inhibited the production of IL‐6 by DCs. However, a 16‐membered macrolide, MDM, did not have any significant effect on these surface markers and cytokine productions. Moreover, AZM increased IL‐10 and CAM decreased IL‐2 productions significantly, when naive T cells derived from spleen were co‐cultured with DCs treated in advance with LPS and these macrolides. These findings suggest that 14‐membered and 15‐membered, but not 16‐membered macrolides play as anti‐inflammatory agents, at least in part, through modulating the functions of DCs. However, each macrolide affects them in different ways.

Clinical features of healthcare-associated pneumonia (HCAP) in a Japanese community hospital: Comparisons among nursing home-acquired pneumonia (NHAP), HCAP other than NHAP, and community-acquired pneumonia

Background and objective:  More than 100 000 Japanese die of pneumonia every year. The number of people residing in nursing homes is increasing with the ageing of the population. In 2005, the American Thoracic Society/Infectious Diseases Society of America (ATS/IDSA) published important guidelines for the management of healthcare‐associated pneumonia (HCAP). In Japan, however, the optimum strategy for management of HCAP is still unclear. The purpose of this study was to clarify the clinical features of patients with HCAP.

Significance of IL‐1β and IL‐1 receptor antagonist (IL‐1Ra) in bronchoalveolar lavage fluid (BALF) in patients with diffuse panbronchiolitis (DPB)

We evaluated the effect of erythromycin therapy on pulmonary function tests and the airway inflammatory response of patients with DPB. The number of neutrophils in BALF obtained from DPB patients was significantly higher than that of healthy volunteers. Treatment with erythromycin (600 mg/day for 12.9 γδ+9.5 months (mean γδ+s.d.)) significantly reduced the total number of cells and neutrophils in the airway, and significantly improved pulmonary function tests. The levels of IL‐1β and IL‐8 were significantly higher in DPB compared with healthy volunteers (P < 0.05, P < 0.05, respectively). IL‐1 Ra in patients is considered to have a weak inhibitory activity for IL‐1β, with approximately five‐fold concentration of IL‐1β compared with that in healthy volunteers (approx. nine‐fold concentration of IL‐1β). Erythromycin therapy significantly reduced these cytokines to levels comparable to those of healthy volunteers, and produced a trend toward reduction in the level of IL‐1Ra in BALF. The level of IL‐1β correlated significantly with the concentration of neutrophils in BALF (r= 0.72, P < 0.01), as well as with the level of IL‐1Ra (r= 0.688, P < 0.05) and IL‐8 (r= 0.653, P < 0.05). A nearly significant or significant correlation was observed between the concentration of neutrophils and levels of IL‐1Ra or IL‐8 in BALF (r= 0.526, P= 0.053 or r= 0.776, P < 0.01, respectively). There was also a significant relationship between FEV, and the concentration of neutrophils in BALF (r= 0.524, P < 0.05). Our results suggest that the relative amounts of IL‐1β and IL‐1Ra or IL‐8 may contribute, at least in part, to the neutrophil‐mediated chronic airway inflammation in patients with chronic airway disease, and long‐term erythromycin therapy may down‐regulate the vigorous cycle between the cytokine network and neutrophil accumulation, with resultant reduction of neutrophil‐mediated inflammatory response.

Inhibitory effect of statins on inflammatory cytokine production from human bronchial epithelial cells.

Statins are 3‐hydroxy‐3‐methylglutaryl‐co‐enzyme A reductase inhibitors of cholesterol biosynthesis, and have been reported to exert pleiotropic effects on cellular signalling and cellular functions involved in inflammation. Recent reports have demonstrated that previous statin therapy reduced the risk of pneumonia or increased survival in patients with community‐acquired pneumonia. However, the precise mechanisms responsible for these effects are unclear. In the present study, we examined the effects of statins on cytokine production from lipopolysaccharide (LPS)‐stimulated human bronchial epithelial cells (BEAS‐2B). Interleukin (IL)‐6 and IL‐8 mRNA expression and protein secretion in LPS‐stimulated cells were inhibited significantly by the lipophilic statin pitavastatin and the hydrophilic statin pravastatin. As these inhibitory effects of statin were negated by adding mevalonate, the anti‐inflammatory effects of statins appear to be exerted via the mevalonic cascade. In addition, the activation levels of Ras homologue gene family A (RhoA) in BEAS‐2B cells cultured with pitavastatin were significantly lower than those without the statin. These results suggest that statins have anti‐inflammatory effects by reducing cytokine production through inhibition of the mevalonic cascade followed by RhoA activation in the lung.

Protective effect of granulocyte colony-stimulating factor (G-CSF) in a granulocytopenic mouse model of Pseudomonas aeruginosa lung infection through enhanced phagocytosis and killing by alveolar macrophages through priming tumour necrosis factor-alpha (TNF-α) production

We investigated the effects of G‐CSF in a granulocytopenic mouse model of Pseudomonas aeruginosa lung infection. The model was prepared by intratracheal instillation of the bacteria, while granulocytopenia was induced by intraperitoneal injection of 4.0 mg of cyclophosphamide (CPA). There was no difference in the survival rate between G‐CSF‐treated animals and the normal group, and the number of neutrophils in the blood and lung recovered to normal in the former group. However, the phagocytic and killing activities of neutrophils were lower in G‐CSF‐treated mice than in controls. Interestingly, the mortality rate increased significantly when anti‐TNF‐α antibody was combined with G‐CSF, although it was intermediate between CPA alone and CPA–G‐CSF‐treated mice. However, the improved mortality was not associated with a change in the number of neutrophils in the circulation and lung. Administration of anti‐TNF‐α antibody resulted in a significant suppression of TNF‐α in bronchoalveolar lavage fluid and of enhanced alveolar macrophage function (phagocytic and bactericidal activity) against P. aeruginosa in G‐CSF‐treated granulocytopenic mice. We showed also increased TNF‐α mRNA expression and TNF‐α production in vitro using G‐CSF‐pretreated alveolar macrophages compared with control untreated macrophages. Our results are the first evidence to suggest that G‐CSF provides a synergistic protective effect against lethal P. aeruginosa lung infection in the granulocytopenic host. This effect is probably due to enhancement of alveolar macrophage function through endogenous TNF‐α production, in addition to increasing the number of circulating neutrophils.

Effect of Erythromycin on Chronic Respiratory Infection Caused by Pseudomonas aeruginosa with Biofilm Formation in an Experimental Murine Model

ABSTRACT Diffuse panbronchiolitis (DPB) is a chronic lower respiratory tract infection commonly associated with persistent late-stage Pseudomonas aeruginosa infection. However, low-dose long-term therapy with certain macrolides is effective in most patients with DPB. The present study was designed to examine the effects of long-term erythromycin (ERY) therapy by using our established murine model of chronic respiratory P. aeruginosa infection. ERY or saline was administered from day 80 after intubation with a P. aeruginosa-precoated tube for the subsequent 10, 20, 40, and 80 days. Bacteriologic and histologic analyses of the murine lungs and electron microscopy of the intubated tube were performed. In the murine model, treatment with ERY for 80 days significantly reduced the number of viable P. aeruginosa organisms in the lungs (P < 0.05). The biofilm formed in situ by P. aeruginosa on the inner wall of the inoculation tube placed into the murine bronchus became significantly thinner after 80 days of ERY treatment. We conclude that the clinical efficacy of macrolides in DPB may be due at least in part to the reduction in P. aeruginosa biofilm formation.

Phenotypic characterization of T cells in bronchoalveolar lavage fluid (BALF) and peripheral blood of patients with diffuse panbronchiolitis; the importance of cytotoxic T cells

We investigated the contribution of T cells in diffuse panbronchiolitis (DPB) by identifying T cell subsets in BALF of 36 patients with DPB, before and after long‐term treatment with macrolide antibiotics, and 16 healthy control subjects. The percentages of lymphocytes and CD3+γδ+ cells in BALF of DPB patients and control subjects were similar, but the absolute number of these cells was higher in DPB patients. Treatment resulted in a significant reduction in the absolute number of these cells. A further two‐colour analysis of T cell subsets in BALF showed a significantly higher ratio and number of CD8+HLA‐DR+ cells in DPB patients. Treatment resulted in a significant reduction of activated T cells. Most BALF CD8+ cells were CD8+CD11b− cytotoxic T cells. The number of these cells in BALF of DPB patients (26.69 ± 5.86 × 103/ml) was higher than the control (2.02 ± 0.38 × 103/ml; P < 0.001), and a significant reduction was observed after treatment (7.69 ± 2.59 × 103/ml; P < 0.01). The number of CD4+ cells was also higher in DPB patients than in controls, and most were CD4+CD29+ memory T cells. However, treatment did not influence the number of these cells. The number of lymphocytes, CD3+γδ+, CD8+CD11b−, CD8+HLA‐DR+, and CD4+CD29+ cells was higher in patients with bacterial infection than in those without bacterial infection, and interestingly, macrolide therapy reduced the number of lymphocytes, CD3+γδ+, CD8+CD11b− and CD8+HLA‐DR+ cells, irrespective of bacterial infection. In peripheral blood, the percentage of CD8+HLA‐DR+ cells was also higher in DPB patients than in healthy subjects, and significantly decreased after treatment. The percentage of CD8+CD11b− cells in peripheral blood was similar in DPB patients and normal subjects, and treatment significantly reduced the percentage of these cells. Finally, the expression of the adhesion molecules CD11a/CD18 (α/β‐chains of LFA‐1) on lung CD3+ cells and CD49d (α‐chain of VLA) on lung CD4+ cells was enhanced compared with that on peripheral blood in DPB patients. Our results suggest that elevation of memory T cells and activation of CD8+ cells, mainly cytotoxic T cells, in the airway lumen of DPB patients may contribute to chronic bronchial inflammation, possibly through up‐regulation of adhesion molecules. Our findings also indicate that macrolide antibiotics may have a direct or indirect suppressive effect on cytotoxic T cells, and as such, reduce inflammation and improve clinical condition.

Elevated levels of interferon γ‐inducible protein‐10 and epithelial neutrophil‐activating peptide‐78 in patients with pulmonary sarcoidosis

Objective and background:  Interferon γ‐inducible protein (IP)‐10 and epithelial neutrophil‐activating peptide (ENA)‐78 belong to the CXC chemokine family and are important factors in inflammatory lung diseases. In sarcoidosis, the potential role of IP‐10 to regulate the migration and activation of T‐cells towards sites of sarcoid activity has been suggested.

In vitro and in vivo potency of polymyxin B against IMP-type metallo-β-lactamase-producing Pseudomonas aeruginosa

Multidrug-resistant Pseudomonas aeruginosa, especially metallo-beta-lactamase (MBL)-producing P. aeruginosa, is an important pathogen in nosocomial infection and emergence of this pathogen has revived interest in polymyxin B (PMB) and colistin (COL). In this study, we evaluated the efficacies of PMB, COL and other antipseudomonal agents against IMP-type MBL-producing P. aeruginosa both in vitro and in vivo. A total of 75 isolates of bla(IMP)-positive P. aeruginosa obtained from clinical specimens (94.6% of isolates demonstrated resistance to beta-lactam, fluoroquinolone and aminoglycoside agents) were evaluated in the in vitro study. More than 90% of the examined isolates were susceptible to PMB (minimum inhibitory concentration for 50/90% of the isolates (MIC(50)/MIC(90)) 4/4 mg/L), although COL was less potent (MIC(50)/MIC(90) 8/16 mg/L). Cyclophosphamide-treated mice were intraperitoneally inoculated with bla(IMP)-positive P. aeruginosa. Treatment with PMB, but not COL, imipenem/cilastatin or aztreonam, significantly improved the survival rate and decreased the number of bacteria in the blood in a dose-dependent manner. Our results indicate that, among the agents studied, PMB is the most effective agent against bla(IMP)-positive P. aeruginosa.