No evidence that rifampicin has glucocorticoid-like immunosuppressive properties leading to suppression of rat-splenocyte proliferation in vitro

J. Visser,*, J. L. Hillebrands and J. Rozing

University of Groningen, Department of Cell Biology, Immunology Section, Building 3215, 11th Floor, A. Deusinglaan 1, 9713 AV Groningen, The Netherlands

Sir,

Rifampicin (RIF) is a widely used antibiotic and is clinically very effective against a variety of organisms, including Streptococcus pneumoniae, Staphylococcus aureus, Haemophilus influenzae and Legionella pneumophila. Furthermore, RIF is also very effective in the treatment of mycobacterial infections.1 In 1998, Calleja et al.2 reported that RIF might act as a non-steroid ligand and activator of the human glucocorticoid receptor. Using reporter gene assays, they demonstrated that RIF could not only induce the transcription of a gene controlled by a glucocorticoid responsive element, but also repressed the transcriptional activation of the IL-2 promotor.2 However, the results of Calleja et al. remain controversial. For example, no glucocorticoid-like side effects of RIF have been reported, although patients have been treated with RIF for >6 months.1 Moreover, Jaffuel et al.3 could not demonstrate an activation of the glucocorticoid receptor in A549 human alveolar cells.

RIF is also widely used to prevent infections in laboratory animals. For example, RIF is commonly used as a prophylaxis to prevent infection of medical devices or biomaterials that are tested in rats.4 Because the glucocorticoid receptors of rats and humans share a strong homology, it is reasonable to hypothesize that RIF might have glucocorticoid-like actions in rats.5 These glucocorticoid-like actions might include suppression of the immune system. This suppression of the immune system by RIF might interfere with results of animal experiments, because the rats might become vulnerable to viral infections or fungal opportunistic infections.

To test whether RIF did have immunosuppressive activity in rats, we investigated whether RIF is capable of suppressing lymphocyte proliferation in vitro. For this purpose splenocytes of Bio-Breeding/Worchester rats were stimulated with 2 mg/L ConA and incubated with RIF or dexamethasone (DEX) in a concentration range from 0.01 to 1 µM. The specific glucocorticoid receptor antagonist RU486 was used to investigate whether the actions of RIF or DEX were mediated via the glucocorticoid receptor.

The figureGo shows the dose–response curve of DEX in the suppression of rat splenocyte proliferation. One micromolar DEX suppressed the splenocyte proliferation by 80%. In contrast, equimolar concentrations of RIF did not result in suppression of proliferation. Accordingly, the suppression of splenocyte proliferation by 1 µM DEX could be completely antagonized by 1 µM RU486. Interestingly, the combination of RU486 and DEX gives a slight, but significant stimulation of splenocyte proliferation. Combination of RIF and RU486 had no effect on the rat splenocyte proliferation.



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Figure.  No evidence that RIF has glucocorticoid-like immunosuppressive properties. Splenocytes of three rats were cultured in vitro in a 96-well flat-bottom culture plate (Nunc, Denmark), at a density of 105 cells/well to a total volume of 200 µL. (a) The cells were stimulated with 2 mg/L ConA (Sigma, St Louis, MO, USA) and dexamethasone ({diamondsuit}; Sigma) or rifampicin ({blacksquare}; Sigma) were added in a concentration range from 0.01 to 1 µM. In (b), 1 µM of RU486 (Roussel Uclaf, Romainville, France) was used to antagonize the effects of 1 µM DEX or 1 µM RIF (symbols: {blacksquare}, ConA; {square}, 1 µM DEX; 1 µM RIF; 1 µM RU486; RU486 + 1 µM DEX; RU486 + 1 µM RIF). Proliferation was measured after 3 days, by the addition of 18.5 kBq (0.5 µCi) methyl-[3H]thymidine with a specific activity of 74 GBq/mmol/well (radiochemical Centre, Amersham, UK) during the last 16 h of culture. Labelled cells were harvested on to glassfibre filters (Canberra Packard, Meriden, CT, USA) and the filters were counted using a liquid scintillation ß-counter (Canberra Packard). The data are expressed as the mean percentage ± s.d. of the proliferation in the absence of DEX, RIF and RU486. The absolute proliferation values in response to ConA were 32 000, 33 600 and 38 330 cpm per individual rat. *P < 0.05 as compared with ConA (Student's t-test).

 
The slight stimulation of the splenocyte proliferation by the combination of RU486 and DEX confirms the previous findings of Wiegers et al.6 In that study, Wiegers et al. demonstrated that occupation of the mineralocorticoid receptor by glucocorticoids stimulated rat lymphocyte proliferation in vitro. The fact that RIF itself does not suppress, or in combination with RU486 enhance, rat splenocyte proliferation in vitro indicates that RIF does not possess glucocorticoid-like activities mediated via the glucocorticoid receptor or the mineralocorticoid receptor.

Taken together, our observations give no indication that RIF under physiological circumstances in vitro has immunosuppressive properties. These results further strengthen the findings of Jaffael et al.3 and Raviglioni et al.,1 that RIF has no glucocorticoid-like actions leading to immunosuppression. Therefore our results suggest that the current practice in the use of RIF in animal experiments need not be changed.

Acknowledgments

The experiments were, according to the national law, approved by the animal experimental committee of the University of Groningen. J.V. and J.L.H. are supported by a grant from the Dutch Diabetes Foundation (DFN98.148, DFN99.028).

Notes

J Antimicrob Chemother 2001; 47: 894–895

* Corresponding author. Tel: +31-50-3632527/2522; Fax: +31-50-3632512; E-mail: j.t.j.visser{at}med.rug.nl Back

References

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2 .  Calleja, C., Passcussi, J. M., Mani, J. C., Maurel, P. & Vilarem, M. J. (1998). The antibiotic rifampicin is a nonsteroidal ligand and activator of the human glucocorticoid receptor. Nature Medicine 4, 92–6.[ISI][Medline]

3 .  Jaffuel, D., Demoly, P., Gougat, C., Mautino, G., Bousquet, J. & Mathieu, M. (1999). Rifampicin is not an activator of the glucocorticoid receptor in A549 human alveolar cells. Molecular Pharmacology 55, 841–6.[Abstract/Free Full Text]

4 .  Van Wijngaerden, E., Peetermans, W. E., Vandersmissen, J., Van Lierde, S., Bobbaewrs, H. & Van Eldere, J. (1999). Foreign body infection: a new rat model for prophylaxis and treatment. Journal of Antimicrobial Chemotherapy 44, 669–74.[Abstract/Free Full Text]

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6 .  Wiegers, G. J., Reul, J. M., Holsboer, F. & de Kloet, E. R. (1994). Enhancement of rat splenic lymphocyte mitogenesis after short term preexposure to corticosteroids in vitro. Endocrinology 135, 2351–7.[Abstract]