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Antimicrobial Agents and Chemotherapy, May 2004, p. 1889-1891, Vol. 48, No. 5
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.5.1889-1891.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Differential Expression Levels of MRP1, MRP4, and MRP5 in Response to Human Immunodeficiency Virus Infection in Human Macrophages

Sylvie Jorajuria,1 Nathalie Dereuddre-Bosquet,2 Karen Naissant-Storck,2 Dominique Dormont,1 and Pascal Clayette2*

CEA, Service de Neurovirologie, Université Paris XI, CRSSA, EPHE, IPSCSPI-BIO, c/o Service de Neurovirologie,1 CEA, Fontenay-aux-Roses, France2

Received 29 July 2003/ Returned for modification 28 October 2003/ Accepted 3 February 2004


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ABSTRACT
 
Multidrug resistance proteins (MRPs) have been reported to be involved in the efflux of some anti-human immunodeficiency virus (HIV) drugs. We show here that MRP1, MRP4, and MRP5 are expressed at the mRNA level in human monocyte-derived macrophages. HIV infection caused increased transcription of these MRPs; however, temporal differences in stimulation are reported.


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TEXT
 
MRP1, MRP4, and MRP5, members of the multidrug resistance protein (MRP) subfamily of ABC transporters, have been reported to transport antiretroviral drugs used to control human immunodeficiency virus (HIV) out of cells in an active process. Protease inhibitors are transported by MRP1 (16), whereas MRP4 and MRP5 transport the phosphorylated derivatives of the nucleoside reverse transcriptase inhibitors zidovudine monophosphate and stavudine monophosphate, respectively (11, 14). These proteins may therefore decrease the efficacy of HIV treatment. However, although MRP1 is present on the surface of cells of the hematopoietic lineage (6, 8), the expression of MRP4 and MRP5 on the surface of the two major HIV target cells, CD4+ T lymphocytes and macrophages, has never been explored. Moreover, little is known about the pathway regulating the expression of these transporters. MRP1 production seems to increase in response to proinflammatory cytokines (7) and to be controlled by the transcription factor Sp-1 (17), which is known to be involved in HIV replication. The high level of HIV protein and infectious-particle production in a CEM cell line overexpressing the gene for MRP1 and infected with HIVIIIB provides further evidence of a relationship between HIV and MRP1 (15). However, the effects of HIV infection or replication on MRP1 expression have not been studied. We also know little of the regulation of MRP4 and MRP5 in this pathophysiological situation, despite the potential importance of this regulation in defining the role of MRPs in therapeutic escape in HIV-infected patients.

We therefore used HIV-1/Ba-L-infected monocyte-derived macrophages (MDM) to investigate (i) changes in the expression of MRP1, MRP4, and MRP5 by quantifying mRNA levels for the mrp1, mrp4, and mrp5 genes and (ii) possible correlations with two major proinflammatory cytokines—tumor necrosis factor alpha (TNF-{alpha}) and interleukin-6 (IL-6)—associated with HIV infection. MDM are useful for such studies because cells of this lineage are involved in the process of inflammation and are the major target of HIV in the central nervous system (CNS). The accessibility of the CNS to anti-HIV compounds may be limited, rendering the CNS a potential viral reservoir. Human MDM were obtained, infected in vitro, and cultured as previously described (12). Viral replication and TNF-{alpha} and IL-6 levels were measured in cell culture supernatants with the RetroSys kit (quantification of reverse transcriptase [RT] activity; Innovagen, Lund, Sweden) and specific enzyme-linked immunosorbent assay (Cayman, Ann Arbor, Mich.). Levels of mRNA for the transporters were determined by noncompetitive RT-PCR using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control (1). The PCR primer sequences were as follows: GAPDH, 5'-ACCACCATGGAGAAGGCTGG-3' and 3'-CTCAGTGTAGCCCAGGATGC-5'; MRP1, 5'-GCACGACCTCCGCTTCAAGA-3' and 3'-TCTTTAGGTCCTCATGCCGCG-5'; MRP4, 5'-TGCCGCTGACGTTTTTAGAT-3' and 3'-TTATAGTCTTTGCGGGTGGTG-5'; MRP5, 5'-GCCCCTTGTCATCCTCTTTT-3' and 3'-AACTCTTGCGTCTCTACTCCAT-5'. The specificity of amplification was confirmed by sequencing of PCR products and sequence alignment and analysis by the Sequed program (Applied Biosystems). PCR assays were performed on two dilutions of each sample and repeated twice. Results are expressed in arbitrary units (AU) as the ratio of the MRP mRNA level to the GAPDH mRNA level. We used the Student unpaired t test (StatView 4.5 software; SAS Institute Inc., Cary, N.C.) to assess changes in the expression levels of MRP mRNA in response to HIV infection and viral replication. Correlations between these mRNA levels and HIV replication or cytokine production were evaluated by the Spearman rank correlation test (StatView 4.5 software).

In uninfected MDM, mRNA levels for the three ABC multidrug transporter genes were low and stable during the 31 days of culture (Fig. 1). As previously reported in long-term cultures of MDM infected in vitro with HIV-1/Ba-L, RT activity increased in culture supernatants, peaking on day 14 after infection (3; data not shown). HIV infection in vitro led to a significant increase in MRP4 expression (3.5 ± 1.5 AU for control cells versus 11.4 ± 1.8 AU for infected cells [P < 0.05]) (Fig. 1A). The induction was transient, with a return to normal levels after the first day (Fig. 1A). In parallel, significant differences in TNF-{alpha} and IL-6 production between uninfected and infected cells (Fig. 2; P < 0.01 for TNF-{alpha} and P < 0.05 for IL-6 [Student t test]) were evidenced at day 1 postinfection. TNF-{alpha} production and induction of mrp4 transcription were found to be correlated 24 h after infection (TNF-{alpha} versus MRP4, P < 0.05 [Spearman's rank correlation test]). On the other hand, no correlation with IL-6 production was observed. MRP1 and MRP5 behaved differently from MRP4 in response to HIV infection and replication. MRP1 and MRP5 displayed similar patterns of mRNA expression (Fig. 1B and C), and infected and uninfected cells expressed the mrp1 and mrp5 genes to similar extents during the first week after infection. In contrast, HIV replication induced a dramatic increase in the MRP1 and MRP5 mRNA levels, coinciding with the peak of viral replication (mrp1 mRNA, 2.6 ± 1.2 AU for uninfected cells versus 17.9 ± 3.5 AU for infected cells, P < 0.01 [Student t test]; mrp5 mRNA, 2.1 ± 0.5 AU for uninfected cells versus 9.5 ± 2.1 AU for infected cells, P < 0.01 [Student t test]). Expression of mrp5 was induced before that of mrp1. The expression of these two transporters was not correlated with the production of cytokines for either TNF-{alpha} or IL-6.



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  		FIG. 1. MRP4 (A), MRP1 (B), and MRP5 (C) mRNA levels in HIV-1/Ba-L-infected ({blacksquare}) and uninfected ({diamond}) MDM. Results are expressed as means ± standard deviations for six culture wells (n = 6). These data were confirmed in a third independent experiment also performed in triplicate. We carried out statistical analysis with the Student unpaired t test. *, P < 0.05; **, P < 0.01.



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  		FIG. 2. TNF-{alpha} (A) and IL-6 (B) production in supernatants of HIV-1/Ba-L-infected ({blacksquare}) and uninfected ({square}) macrophages. The expression of these cytokines was measured at days 1 and 11 postinfection. Results are expressed as means ± standard deviations for nine culture wells (n = 9). We carried out statistical analysis with the Student unpaired t test. *, P < 0.05; **, P < 0.01.

MRP4 and MRP5 have similar topologies (2) and substrate affinities (13). We detected the transcripts for both of these transporters in human macrophages. Despite these similarities, MRP4 an MRP5 behaved very differently in response to HIV infection and replication. As described for the P-glycoprotein gene (Jorajuria et al., submitted for publication), transcription of mrp4 was induced by HIV-cell contact and coincided with TNF-{alpha} production, suggesting that the preintegration steps of the HIV biological cycle and proinflammatory cytokines may account for this upregulation. The mrp4 promoter has not yet been fully characterized in terms of transcription factor binding sites. However, it seems likely that some of the binding sites present in this promoter may also be present in the promoter of MDR1, which encodes the P glycoprotein, but absent from the promoters of mrp1 and mrp5, and vice versa. For instance, a binding site for Ets1, which is involved in regulation of the immune response to viral infections (5), is present in mrp1 and mrp5 promoters but much less common in MDR1 and mrp4 promoters (determined from reference 18). Ets1, by binding to specific sites in the U3 region of the HIV-1 long terminal repeat (4), stimulates HIV transcription and replication (10). It is therefore not surprising that the expression of mrp1 and mrp5 increases with HIV replication. Our results for MRP1 are consistent with those of Speck et al. (15) but conflict with those of Meaden et al. (9), who reported no change in MRP1 levels at the surface of peripheral blood mononuclear cells in HIV-infected patients. However, Meaden et al. evaluated MRP1 levels in patients who had received highly active antiretroviral therapy, and this antiretroviral treatment may have affected MRP1. Interestingly, the induction of mrp1 and mrp5 transcription coincided with maximal HIV production and was not correlated with cytokine production. This suggests that the transcription of these two genes and HIV production may involve similar regulation mechanisms.

In conclusion, this study is the first to provide insight into the expression of MRP4 and MRP5 in human macrophages and differences in the regulation of expression of MRP transporters. The molecular basis of the observed differences in response to HIV are unclear, but our results suggest that HIV may favor the efflux of antiretroviral drugs in macrophages, thereby decreasing their pharmacological effects, particularly in organs such as those of the CNS.


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ACKNOWLEDGMENTS
 
We thank the Centre de Transfusion Sanguine des Armées (CTSA, Hôpital Percy, Clamart, France) and Julie Sappa for excellent help in preparation of the manuscript.

This work was supported by Agence Nationale de Recherche sur le SIDA (ANRS, Paris, France) and by the Commissariat à l'Energie Atomique (CEA, Fontenay aux Roses, France). S.J. received a grant allocated by ANRS.


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FOOTNOTES
 
* Corresponding author. Mailing address: SPI-BIO c/o Service de Neurovirologie, CEA, DSV/DRM, 18, route du Panorama, B.P. 6, 92265 Fontenay-aux-Roses Cedex, France. Phone: 33 (0)1 46 54 87 69. Fax: 33 (0)1 46 54 77 26. E-mail: clayette{at}dsvidf.cea.fr. Back


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Antimicrobial Agents and Chemotherapy, May 2004, p. 1889-1891, Vol. 48, No. 5
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.5.1889-1891.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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