1 EA 3925, Laboratoire de Bactériologie-Hygiène, Hôpital Calmette, Boulevard du Professeur J. Leclercq, 59037 Lille, France; 2 Centre de Ressources et de Compétences pour la Mucoviscidose, Clinique de Pédiatrie, Hôpital Jeanne de Flandre, 59037 Lille, France; 3 Centre de Ressources et de Compétences pour la Mucoviscidose Adultes, Hôpital Calmette, and Inserm U416, Institut Pasteur de Lille, 59800 Lille, France
Received 2 February 2005; returned 22 February 2005; revised 15 March 2005; accepted 28 April 2005
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Abstract |
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Methods: To establish this, we initiated a prospective study to measure the concentration of the three pro-inflammatory cytokines IL-8, IL-6 and TNF- in the sputum from 20 cystic fibrosis (CF) patients (15 teenagers and 5 young adults) during cycles and off cycles.
Results: A significant decrease in IL-8 (P = 0.001) and a more moderate decrease in IL-6 (P = 0.046) and TNF- (P = 0.052) levels were observed during cycles, even if no significant decrease in the number of leucocytes was observed.
Conclusions: These results associated with a decrease in the Pseudomonas aeruginosa population can contribute in part to the beneficial effect of intermittent inhaled tobramycin on pulmonary function.
Keywords:
Pseudomonas aeruginosa
,
IL-8
,
IL-6
,
TNF-
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Introduction |
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To improve lung function, intermittent inhaled administration of tobramycin has been proposed for the delivery of very high local concentrations and optimal antipseudomonal activity.3 However, very little information is available on the effect of this therapy on the inflammatory process in CF patients. Treatment consists of twice daily inhalation of 300 mg of tobramycin during a 28 day treatment period (on cycle) followed by a 28 day no-treatment period (off cycle). This study was a prospective analysis designed to measure the concentrations of three pro-inflammatory cytokines (IL-8, IL-6 and TNF-) in the sputum of P. aeruginosa-infected CF patients treated with intermittent tobramycin aerosols.
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Patients and methods |
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Fifteen teenagers [age (years): mean, 15.8; range, 13.518.8] and 5 young adults [age (years): mean, 23.1; range 21.125.9] followed at the Lille University Hospital Pediatric and Adult Cystic Fibrosis Centers were included. All of them were colonized with P. aeruginosa and received at least three cycles of inhaled tobramycin before the study. Treatment with oral and/or intravenous antipseudomonal antibiotics within 4 weeks preceding the study was an exclusion criterion. Written informed consent was obtained from all patients (and/or family).
Methods
Sputum samples were collected for each patient after spontaneous expectoration at the end of antibiotic aerosol cycles and at the end of off cycles, and were processed within 2 h. An aliquot of homogenized sputum in fresh 10% (v/v) dithiothreitol was used for quantitative bacterial cultures. To check the quality of samples, total cell counting was carried out in a haemocytometer, and cell viability was evaluated by means of the Trypan Blue exclusion method. A quantity of 106 cells/mL including less than 20% of squamous epithelial cells and a cell viability superior to 80% were retained as quality criteria for cytokine measurement.
TNF-, IL-8 and IL-6 concentrations were measured using commercially available enzyme-linked immunosorbent assay kits (R&D systems, Abingdon, UK) according to the recommended protocols. They were quantified by comparison with a standard curve generated using the appropriate recombinant human cytokine. Sensitivity was as follows: IL-8, 10 ng/mL; IL-6, 0.7 pg/mL; and TNF-
, 4.4 pg/mL.
Statistical analysis
Results obtained for each patient during antibiotic cycles and off cycles were compared using the Wilcoxon test. Differences were considered significant at P < 0.05.
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Results |
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Discussion |
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Reduction of pro-inflammatory cytokine secretion affects essentially IL-8 and more moderately IL-6 and TNF-. Since the host inflammatory response is usually correlated with pulmonary infection, the significant decrease in the P. aeruginosa population may explain the beneficial effect of inhaled tobramycin on inflammation. These results are similar to those described after intravenous therapy.6,7 Along this line, it is known that CF cell lines produce significant amounts of pro-inflammatory cytokines in response to P. aeruginosa infection.8 However, one cannot exclude the possibility that inhaled tobramycin, which inhibits the production of IL-8 more than that of IL-6 and TNF-
, has a direct action on CF epithelial cells for which an up-regulation of nuclear factor-
B (NF-
B) leading especially to an activation of IL-8 production has been demonstrated.9
In conclusion, tobramycin aerosol treatment results in a marked reduction in IL-8 and IL-6 sputum levels which may contribute in part to the beneficial effect of this treatment on pulmonary function.
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Acknowledgements |
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References |
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DiMango E, Ratner AJ, Bryan R et al. Activation of NF-kappaB by adherent Pseudomonas aeruginosa in normal and cystic fibrosis respiratory epithelial cells. J Clin Invest 1998; 101: 2598605.
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