a Infectious Diseases and Microbiology, Charing Cross Campus, Imperial College; b Cytokine Biology and Signal Transduction Laboratory, Kennedy Institute of Rheumatology, London, UK
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Abstract |
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Introduction |
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The Mycobacterium ulcerans toxin has been shown to be immunosuppressive and block NF-B activation by tumour necrosis factor (TNF).4 As the structure of M. ulcerans toxin, a polyketide,5 is related to that of rifamides we decided to investigate whether these drugs inhibited NF-
B activation, particularly in response to TNF, a cytokine important to the host defence against bacterial infection.6 We now show that rifamides can inhibit TNF-induced NF-
B activation, a result that may help explain the immunosuppressive properties of these drugs.
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Materials and methods |
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The M. ulcerans pathogenic strain (7634) from an original clinical isolate was provided by Dr J. L. Stanford (Middlesex Hospital, London, UK). Mycobacteria were grown in Dubos broth base medium (Difco, Oxford, UK) as previously described.4 Rifamides were obtained from Sigma (Poole, UK), or were given by Marion-Roussel, Romainville, France (rifapentine) and Pharmacia, Milton Keynes, UK (rifabutin).
Preparation of M. ulcerans culture filtrate (CF) and ethyl acetate extracted filtrate (EAEF)
Culture filtrates (CFs) were prepared as described by Pahlevan et al.4 CF was then extracted twice with ethyl acetate. The organic phase was collected and concentrated by evaporation, and hence termed ethyl acetate extracted filtrate (EAEF). EAEF had the same activities as our previously partially purified M. ulcerans toxin, aHDL,4 or a chemically defined extracted factor from M. ulcerans CF termed MUPT (M. ulcerans polyketide toxin) (results not shown).4,7
Cell culture and assay for ß-galactosidase
The human T cell line Jurkat subclone 3KB5.2 (NF-B) containing the NF-
B ß-galactosidase reporter gene (given by Prof. Herzenberg, Stanford, CA, USA) was maintained as described previously.4 The cells were washed twice and resuspended at 2 x 106 cells/mL in complete RPMI, without phenol red. One hundred microlitres per well of cell suspension were added to a 96-well plate and treated in the presence or absence of compounds with stimuli: TNF (20 ng/mL) or phorbol myristate acetate (PMA) (50 ng/mL) in triplicate overnight. The cells were lysed and ß-galactosidase activity was measured as described previously.4
Electrophoretic mobility shift assay (EMSA)
Following activation of cells with TNF or PMA for 30 min, cells were lysed and nuclear proteins were extracted and assayed for NF-B DNA binding activity using an oligonucleotide encoding the NF-
B binding sequence as described previously.4
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Results and discussion |
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Intracellular concentrations of rifamides other than rifampicin may be much higher than serum levels,9,10 therefore, the highest concentration tested in our experiments may prove clinically significant and more likely to be immunosuppressive. This effect may impinge on the management of tuberculosis and is, perhaps, especially significant in patients with concurrent AIDS, where additional immunosuppression may cause reoccurrence (see preliminary findings reviewed by Corbett et al.1). There have been relapses reported when rifapentine was used to treat tuberculosis in HIV patients.10 It is possible that this rifamide has an immunosuppressive effect, which by encouraging recurrences promotes the development of rifamide monoresistance in these patients. Although the rifamides share with the M. ulcerans toxin the ability to inhibit NF-B activity, the data would indicate that the mechanisms by which they act are distinct, possibly linked to the different side chain structures. Such structural differences may also explain our observation that rifamides, unlike M. ulcerans toxin,4 were unable to block LPS-induced TNF production from human peripheral blood monocytes (data not shown). The result would discount another potential mechanism of rifamide immunosuppression.
In summary, these data show that rifamides have the potential to block NF-B activation by TNF, which could provide a mechanism for the immunosuppressive properties of these drugs.
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Acknowledgements |
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Notes |
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References |
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Pahlevan, A. A., Wright, D. J., Andrews, C., George, K. M., Small, P. L. & Foxwell, B. M. (1999). The inhibitory action of Mycobacterium ulcerans soluble factor on monocyte/T cell cytokine production and NF-B function. Journal of Immunology 163, 392835.
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George, K. M., Chatterjee, D., Gunawardana, G., Welty, D., Hayman, J., Lee, R. et al. (1999). Mycolactone: a polyketide toxin from Mycobacterium ulcerans required for virulence. Science 283, 8547.
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Nunez Martinez, O., Ripoll Noiseux, C., Carneros Martin, J. A., Gonzalez Lara, V. & Gregorio Maranon, H. G. (2001). Reactivation tuberculosis in a patient with anti-TNF- treatment. American Journal of Gastroenterology 96, 16656.[ISI][Medline]
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George, K. M., Barker, L. P., Welty, D. M. & Small, P. L. (1998). Partial purification and characterization of biological effects of a lipid toxin produced by Mycobacterium ulcerans. Infection and Immunity 66, 58793.
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9 . Parenti, F. & Lancini, G. (1997). Rifamycins. In Antibiotics and Chemotherapy; Anti-infective Agents and their Use in Therapy, (O'Grady, F., Lambert, H. P., Finch, T. G. & Greenwood, D., Eds), pp. 4539. Churchill Livingstone, New York.
10 . Vernon, A., Burman, W., Benator, D., Khan, A. & Bozeman, L. (1999). Acquired rifamycin monoresistance in patients with HIV-related tuberculosis treated with once-weekly rifapentine and isoniazid. Tuberculosis Trials Consortium. Lancet 353, 18437.[ISI][Medline]
Received 18 October 2001; returned 27 November 2001; revised 10 December 2001; accepted 10 December 2001