a Departments of Biochemistry and b Medicine, University of Texas Health Center at Tyler, Tyler, 11937 US Highway 271, TX 75708-3154, USA
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Abstract |
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Introduction |
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Our preliminary studies showed that administration of azithromycin (a macrolide antibiotic) and clarithromycin (an azalide antibiotic closely related to the macrolide group) to patients with mycobacterial infections increases plasma IL-8 concentrations. Therefore, in the present study we examined the effect of azithromycin and clarithromycin on IL-8 production by human white blood cells. We also explored their effect on human lung macrophages because azithromycin and clarithromycin accumulate primarily in lungs, and because both azithromycin and clarithromycin have been specifically detected in alveolar macrophages.3,4
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Materials and methods |
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All studies involving human blood (informed written consent was obtained from all the subjects) and lung macrophages were approved by the Human Subjects Investigation Committee of the University of Texas Health Center at Tyler.
Reagents
Azithromycin was provided by Pfizer Inc. (New York, NY, USA) and clarithromycin by Abbott Laboratories (North Chicago, IL, USA). For the stimulation of human white blood cells, the antibiotics were either extracted from tablets with phosphate-buffered saline (PBS) to obtain a 10 mg/mL solution, or pure preparations were dissolved in methanol (4 mg/mL), and then diluted in lipopolysaccharide (LPS)-free PBS (Sigma Chemical Co., St Louis, MO, USA). Alveolar macrophages were stimulated with the pure antibiotic preparations dissolved in methanol.
Whole blood stimulation
Blood samples were taken from six healthy donors. Aliquots of blood were incubated with different concentrations of azithromycin (0.0440 mg/L), clarithromycin (0.04 40 mg/L), LPS (10 mg/L; positive control) or LPS-free PBS (negative control) overnight at 37°C with gentle shaking. After 24 h, plasma was separated from cells. The cells were then processed according to the modified method of Darbonne et al.5 Briefly, after incubation with a lysing buffer (0.85% NH4Cl/10 mM TrisHCl pH 7.5 containing 0.25% bovine serum albumin) to disrupt red blood cells, the samples were centrifuged. Supernatants containing the lysed red blood cells were treated with Triton X-100 to remove cell membranes.
Stimulation of alveolar macrophages
Alveolar macrophages are routinely isolated in our laboratory from lungs removed surgically from patients with lung cancer as described previously.6 A white cell differential count was performed, and only those preparations containing <5% neutrophils were used. Cell cultures were maintained in RPMI-1640 medium (Gibco-BRL, Grand Island, NY, USA). Macrophages were incubated with different concentrations of azithromycin (0.41000 mg/L), clarithromycin (0.41000 mg/L), LPS (10 mg/L; positive control) or LPS-free PBS (negative control) overnight at 37°C. Then, the media were collected and stored for further analysis. The number of viable cells was estimated using a tetrazolium salt, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] as described previously.7
IL-8 ELISA
The concentration of IL-8 was measured in plasma, red cell lysates and macrophage media in an ELISA assay using a matched antibody pair according to the manufacturer's protocol (R&D Systems, Minneapolis, MN, USA). Briefly, 96-well microtitre plates were coated with monoclonal anti-human IL-8 antibody. After blocking, the plates were incubated with samples overnight. Then, the plates were washed and incubated with biotinylated goat anti-human IL-8 polyclonal antibody followed by streptavidinperoxidase (Zymed Laboratories, Inc., San Francisco, CA, USA). The plates were developed with tetramethyl benzidine (Sigma).
Statistical analysis
Differences between groups were analysed by a simple one-way analysis of variance (ANOVA). All statistics were performed using SIGMASTAT (SPSS Science Inc., Chicago, IL, USA).
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Results and discussion |
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IL-8 production was increased in a similar manner when the blood was incubated with pure azithromycin and clarithromycin (0.0440 mg/L) (Figure 1a and b). Both plasma and red blood cell-associated IL-8 were induced as a result of the stimulation (P < 0.05 compared with control) (Figure 1a and b
). The amount of IL-8 associated with red blood cells was significantly lower than soluble IL-8 (P < 0.05), and azithromycin and clarithromycin were less effective in stimulating IL-8 production than LPS (P < 0.05) (Figure 1a and b
). Appropriate dilutions of methanol alone did not affect IL-8 production.
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It is important to note that other cell types may also be affected by azithromycin and clarithromycin. Takizawa et al.,10 for example, reported that clarithromycin suppressed IL-8 production by human bronchial epithelial cells but was not cytotoxic for these cells. In summary, our findings indicate that azithromycin and clarithromycin could modify IL-8 function in mycobacterial infections, which may partially explain their efficacy in these infections. Further work is needed to establish what mediators are involved in modulating IL-8 production.
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Acknowledgments |
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Notes |
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References |
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2
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Yamazaki, Y., Kubo, K., Sekiguchi, M. & Honda, T. (1998). Analysis of BAL fluid in M. avium-intracellulare infection in individuals without predisposing lung disease. European Respiratory Journal 11, 122731.
3 . Patel, K. B., Xuan, D., Tessier, P. R., Russomanno, J. H., Quintiliani, R. & Nightingale, C. H. (1996). Comparison of bronchopulmonary pharmacokinetics of clarithromycin and azithromycin. Antimicrobial Agents and Chemotherapy 40, 23759.[Abstract]
4 . Rodvold, K. A., Gotfried, M. H., Danziger, L. H. & Servi, R. J. (1997). Intrapulmonary steady-state concentrations of clarithromycin and azithromycin in healthy adult volunteers. Antimicrobial Agents and Chemotherapy 41, 1399402.[Abstract]
5 . Darbonne, W. C., Rice, G. C., Mohler, M. A., Apple, T., Hebert, C. A., Valente, A. J. et al. (1991). Red blood cells are a sink for interleukin 8, a leukocyte chemotaxin. Journal of Clinical Investigation88, 13629.
6 . Miller, E. J., MacArthur, C. K., Gray, L. D. & Cohen, A. B. (1990). Liberation of a neutrophil enzyme-releasing peptide from the surface of human alveolar macrophages. American Journal of Physiology 258, 32833.
7 . Denizot, F. & Lang, R. (1986). Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. Journal of Immunological Methods 89, 2717.[ISI][Medline]
8 . Friedland, J. S., Hartley, J. C., Hartley, C. G., Shattock, R. J. & Griffin, G. E. (1995). Inhibition of ex vivo proinflammatory cytokine secretion in fatal Mycobacterium tuberculosis infection. Clinical and Experimental Immunology 100, 2338.[ISI][Medline]
9 . Sangari, F. J., Parker, A. & Bermudez, L. E. (1999). Mycobacterium avium interaction with macrophages and intestinal epithelial cells. Frontiers in Bioscience 4, 5828.
10
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Takizawa, H., Desaki, M., Ohtoshi, T., Kawasaki, S., Kohyama, T., Sato, M. et al. (1997). Erythromycin modulates IL-8 expression in normal and inflamed human bronchial epithelial cells. American Journal of Respiratory and Critical Care Medicine 156, 26671.
Received 15 August 2000; returned 3 November 2000; revised 19 December 2000; accepted 22 January 2001