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International Immunology Advance Access originally published online on September 28, 2006
International Immunology 2006 18(11):1603-1606; doi:10.1093/intimm/dxl094
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© The Japanese Society for Immunology. 2006. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Multidrug-resistance-associated protein 1 (Mrp1) is probably not required for murine Th cell activation

Peter Kleemann1, Baerbel Casper1, Magdalena Huber1, John D. Schuetz2 and Michael Lohoff1

1 Institut fuer Medizinische Mikrobiologie und Krankenhaushygiene, University of Marburg, BMFZ, Hans-Meerweinstrasse, 35043 Marburg, Germany
2 Department of Pharmaceutical Sciences, St Jude Children's Research Hospital, Memphis, TN, USA

Correspondence to: P. Kleemann; E-mail: kleemann{at}med.uni-marburg.de


   Abstract
Top
 Abstract
Introduction
 Methods
 Results and Discussion
 References
 
Previously, we have demonstrated that multidrug-resistance-associated protein 1 (Mrp1) represents an activation marker for murine Th1 cells and is constitutively expressed by Th2 cells. Using the inhibitor MK571, we and others also suggested that Mrp1 is necessary for Th cell activation. However, herein, we show that Mrp1-deficient Th cells can be differentiated to a similar extent to Th1 and Th2 cells in vitro and, upon re-stimulation, produce comparable amounts of IL-2, IFN{gamma} and IL-4. Mrp1-deficient mice are equally susceptible than wild-type mice to infection with the protozoan parasite Leishmania major, a well-respected model for in vivo Th1 and Th2 cell differentiation. Intriguingly, MK571 is able to completely block activation of Mrp1-deficient Th cells. Most likely, therefore, the molecule relevant for Th cell activation which is blocked by MK571 is different from Mrp1. While these results are compatible with our previously reported data on Mrp1 expression, they contradict our previous conclusions about Mrp1 function in murine Th1 cells as well as those published in a very recent report in this journal on human Th cells.

Keywords: ABC transporter, MK571, MRP, Th cell, stimulation


   Introduction
Top
 Abstract
 Introduction
Methods
 Results and Discussion
 References
 
The ATP-binding cassette (ABC) transporter molecule Mrp1 was originally detected by virtue of its ability to extrude cytotoxic drugs from lung tumor cells (1). We have previously shown that this molecule is constitutively expressed by cloned Th2 cells, while it is absent on resting Th1 cells (2). Th1 cells acquire Mrp1 upon stimulation (2, 3). Using the fluorescent anion fluo-3 as a target for Mrp1, we showed that Mrp1 is a powerful marker for parasite-specific activated Th cells in vivo in the model system of murine leishmaniasis (2). This protozoan parasite infects macrophages and causes a lethal infection with expansion of Th2 cells in susceptible (e.g. BALB/c) mice, while resistant mice (e.g. FVB, C57BL/6) mount a Th1 response with production of IFN{gamma} and spontaneously clear the infection (4).

Many of the studies on Mrp1 have made use of the reagent MK571, considered to be specific for Mrp1. Several years ago, we demonstrated that MK571 is able to completely block activation of Th cells and, from this set of data, we concluded at the time that Mrp1 is relevant for murine Th cell activation (3). Very recently, a report appeared in this journal that described a blocking activity of MK571 on activation of human Th cells (5). Again, this activity was attributed to MRP1, the human homologue of murine Mrp1.

Given these published conclusions on the relevance of Mrp1 and MRP1 for Th cell activation, we decided to publish a set of ‘negative’ data accumulated in the last 3 years in our laboratory by using Mrp1-deficient mice. We show that Th cells from these mice are absolutely normal in their activation characteristics and produce normal levels of cytokines in vitro. Likewise, the course of leishmaniasis in these mice, both in the susceptible and the resistant genetic background, is normal. Interestingly, activation of Mrp1-deficient Th cells can be blocked by MK571 to the same extent as that of wild-type Th cells arguing for a target molecule of MK571 which is different from Mrp1.


   Methods
Top
 Abstract
 Introduction
 Methods
Results and Discussion
 References
 
Mice
Mrp1-deficient (mrp1–/–) mice of the FVB background were kindly provided by J. Wijnholds, Netherlands Cancer Institute, Amsterdam (6). In our animal facility, these mice were backcrossed for six generations to the BALB/c background. In the experiments, female mrp1–/– mice or mrp1+/– littermate controls were used at the age of 6–10 weeks.

Murine leishmaniasis
Mice were infected in the right hind footpad with Leishmania major promastigotes and the infection was monitored by measuring the lesion size, as described previously (7). Determination of parasite burden and antigen-specific re-stimulation of Th cells from the lesion-draining lymph nodes in vitro were also performed according to published methods (7). The cytokine contents in 48-h culture supernatants were measured by ELISA using reagents from Becton-Dickinson (BD).

Th cell stimulation and staining for Mrp1
Naive CD4+ T cells from FVB mice were purified using the MACS system, as described (8). These cells were differentiated in Th1 or Th2 cells and re-stimulated in vitro, as reported (8). At variance to the published protocol, all primary stimulations were performed in the presence of anti-CD28 (clone 37.51) at a concentration of 2.5 µg ml–1. Culture supernatants were harvested 24 h after re-stimulation and the amounts of IL-2, IL-4 and IFN{gamma} were tested by ELISA using reagents from BD. Expression of the Th cell activation markers CD44 and CD69 was measured by flow cytometry with directly conjugated antibodies from BD 24 and 48 h after primary stimulation using the FACScan and the CellQuest software (BD). In some experiments, culture supernatants were harvested and tested 48 h after primary stimulation for the presence of IL-2. Also, some cultures received the compound MK571 (Alexis, Gruenberg, Germany) at concentrations indicated in Fig. 5. In the experiment described in Fig. 1, CD4+ T cells were stained for Mrp1 24 h after re-stimulation. To do this, the cells were fixed with PFA for 20 min, permeabilized by 0.5% saponin and stained using anti-Mrp1 antibody (1 µg ml–1, kindly providid by R. J. Scheper, Free University Hospital, Amsterdam, The Netherlands) and PE-conjugated goat anti-rat antibodies (10 µg ml–1, Dianova, Hamburg, Germany) followed by FACS analysis.


Figure 5
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  		Fig. 5 MK571 blocks activation of mrp1–/– Th cells. Naive CD4+ Th cells (1 x 106 ml–1) were cultured with or without immobilized anti-CD3 (5 µg ml–1) and soluble anti-CD28 (2.5 µg ml–1) in the presence or absence of the depicted amounts of MK571. After 48 h, culture supernatants were harvested and tested for the presence of IL-2 (filled bars) or IFN{gamma} (open bars) by ELISA. Bars denote the SD of the ELISA. n.d.: not detectable. Three experiments were performed with similar outcome.

 

Figure 1
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  		Fig. 1 The mrp1–/– mice used are truly deficient for Mrp1. Naive CD4+ Th cells (1 x 106 ml–1) were cultured with or without immobilized anti-CD3 (5 µg ml–1) and soluble anti-CD28 (2.5 µg ml–1) for 72 h. After a resting phase in the absence of anti-CD3, but presence of IL-2, the cells were washed and re-stimulated (1 x 106 ml–1) by immobilized anti-CD3 (5 µg ml–1). Twenty-four hours later, the cells were fixed, permeabilized and stained for expression of Mrp1 followed by FACS analysis.

 

   Results and Discussion
Top
 Abstract
 Introduction
 Methods
 Results and Discussion
References
 
We have previously shown that Mrp1 represents an activation marker for Th1 cells, while cloned Th2 cells express Mrp1 constitutively (2). MK571 was able to block activation of Th1 cells which, at the time of the publication, led us to conclude that Mrp1 is required for Th1 cell activation.

To confirm the relevance of Mrp1 for Th cell activation, we used the in vivo model of leishmaniasis in mice deficient for Mrp1. The lack of Mrp1 was confirmed by deficient intracellular staining for Mrp1 in activated Th cells of these mice as compared with Th cells from mrp1+/– littermates (Fig. 1). Surprisingly, the course of leishmaniasis did not differ between these mice and control gene-competent littermates, as measured by development of lesion size in the infected footpad. This was true for the resistant phenotype in mrp1–/– FVB mice as well as for the susceptible phenotype in mrp1–/– BALB/c mice (Fig. 2). Like the lesion size, the parasite burden in the lesion-draining lymph nodes of these mice was unaltered with equally high burdens in lymph nodes of mrp1–/– and mrp1+/– BALB/c mice and equally low burdens in lymph nodes of mrp1–/– and mrp1+/– FVB mice (data not shown). Similar data were obtained when the parasite burden was tested 2 or 7 weeks after infection or when the infection was initiated using 1 x 104 instead of 2 x 107 leishmania promastigotes (data not shown).


Figure 2
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  		Fig. 2 Course of leishmaniasis in mrp1–/– mice. Mice of the BALB/c or FVB genetic backgrounds (four mice per group) and of the depicted Mrp1 status (–/– or +/–) were infected with 2 x 107 Leishmania major promastigotes in the right hind footpad. As a parameter for lesion development, the relative increase in footpad thickness as compared with the non-infected contralateral footpad is shown. BALB/c mice were euthanized 5 weeks after infection due to necrosis of the lesion. Bars denote the SD of the increase in footpad thickness in each group. Two experiments were performed with similar results.

 
At different times after infection, single-cell preparations of the draining lymph nodes were re-stimulated in vitro with leishmania antigens and the cytokines produced were measured by ELISA. As shown for FVB mice 7 weeks after infection, cytokine production was dependent on the presence of leishmania antigens and followed the expected Th1 cytokine pattern (high levels of IFN{gamma} but not IL-4, Fig. 3A). In contrast, cytokines of a Th2 pattern were secreted by cells from infected BALB/c mice 5 weeks after infection and, again, the amounts of cytokines did not differ between cells from mrp1–/– and mrp1+/– mice (Fig. 3B). As for BALB/c-derived cells, no difference in cytokine production was noted in the presence or absence of exogenous leishmania antigens. This is a common finding in this mouse strain and can be attributed to the high parasite loads present in the lymph node preparations. Similar data were obtained when lymph node cells were tested 2 weeks after infection (data not shown).


Figure 3
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  		Fig. 3 Cytokine production in leishmania-infected mice. Mice [(A) FVB and (B) BALB/c] were infected, as described for Fig. 2. Single-cell suspensions of the draining popliteal lymph nodes were prepared 7 weeks (FVB) or 5 weeks (BALB/c) after infection. Cells (2 x 105) were co-cultured with 3 x 105 syngeneic-irradiated (20 Gy) spleen cells from normal mice with (+Ag) or without (–Ag) leishmania antigens (3 x 105 promastigote equivalents) for 48 h in triplicates and supernatants were tested for the depicted cytokines by ELISA. Each mouse was analyzed separately and the bars denote the SD for the different mice in one group. Two experiments were performed with similar results.

 
In light of our previously published findings, these results were unexpected. To evaluate the role of Mrp1 for Th cell function independently, we next used a well established protocol to differentiate naive CD4+ Th cells in Th1 or Th2 cells in vitro using anti-CD3 in conjunction with anti-CD28 and IL-12 plus anti-IL-4 or IL-4 plus anti-IFN{gamma}. After a resting period, the cells were re-stimulated by anti-CD3 and cytokines produced were measured by ELISA. As expected, IFN{gamma} but not IL-4 was produced by Th1-driven cells (Fig. 4), while the reverse was true for Th2-driven cells. Again, no significant difference was noted between cells from mrp1–/– and mrp1+/– mice.


Figure 4
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  		Fig. 4 In vitro differentiation of Mrp1-deficient Th cells. Naive CD4+ T cells were purified and shifted in Th1 or Th2 cells for 3 days, as described in Methods, rested for 72 h in the presence of IL-2 (1 ng ml–1), re-stimulated by solid-phase coupled anti-CD3 (5 µg ml–1) for 24 h and supernatants were tested for the presence of cytokines. Bars denote the SD of the ELISA. n.d.: not detectable. Three experiments were performed with similar outcome.

 
In our previous publication published several years ago as well as in a very recent publication published in this journal about human Th cells, a blocking activity of the compound MK571 was used to conclude that Mrp1 or the human homologue MRP1 is relevant for Th cell activation (3, 5). This conclusion is based on the assumption that MK571 is specific for Mrp1/MRP1. However, the results in the current publication strongly argue against a decisive role of Mrp1 for murine Th cell activation. Therefore, in the final set of experiments, we addressed the question whether MK571 would have a blocking capacity on the activation of mrp1–/– Th cells. The results of a representative experiment are shown in Fig. 5: Naive CD4+ mrp1–/– Th cells were stimulated for 48 h by anti-CD3 and anti-CD28 in the presence or absence of MK571. Thereafter, IL-2 and IFN{gamma} production was tested in the culture supernatants. As can be seen, IL-2 and IFN{gamma} production was strictly dependent on anti-CD3/28. MK571 blocked IL-2 as well as IFN{gamma} secretion dose dependently. Remarkably, the amounts of MK571 necessary for suppression were exactly the same as those reported for murine mrp1+/+ or human MRP1+/+ Th cells.

From this data, we would like to conclude that in contrast to previous conclusions, Mrp1 plays no major role for Th cell activation and that MK571 likely exerts its effects by targeting a molecule different from Mrp1. Of note, since our original analysis of Mrp1 function on Th cells, Mrp molecules have increased in number and now form a whole family of molecules in mice and humans (9). It has already been shown that MK571 also suppresses the activities of Mrp4 and Mrp5 (10). In addition, MK571 is also known to inhibit the ABC transporter abcg2 and the leukotriene D4 receptor (LTD4R) (11, 12). As for Mrp4, we have preliminary evidence using mrp4–/– mice, that Mrp4 is also irrelevant for Th cell activation. In addition, we have used several other LTD4R antagonists and did not observe any effects of these compounds on Th cell activation. In conclusion, therefore, the molecular target which is used by MK571 to inhibit Th cell activation is still unknown and, quite likely, may represent a different member of the Mrp family. Despite this, our previous results on Mrp1 expression in Th cells and the role of Mrp1 as a marker for in vivo-activated Th cells (2) are still valid.

Our data challenge conclusions of the above cited paper by Zhang et al. (5) of a relevance of MRP1 for human Th cell function. Of course, we cannot exclude the possibility that human Th cells behave differently than murine Th cells in this regard or, alternatively, that our treatment protocol with anti-CD3/28 has somehow different effects than stimulation by superantigens, as performed by Zhang et al. (5). Also, it should be mentioned that in other complex in vivo models, like dextran sulphate sodium-induced colitis, dendritic cell migration or drug-induced ear edema, mrp1–/– mice had a phenotype (6, 13, 14). However, in all these models, a major contribution to the respective model stems from cells other than Th cells. Thus, although the results obtained in these models may allow for the assumption of a subtle effect of Mrp1 in Th cells, they certainly do not prove it.


   Acknowledgements
 
We thank Scheper, Amsterdam, Netherlands, for the gift of anti-Mrp1 antibody. This work was supported by grant LO396/1-4 from the Deutsche Forschungsgemeinschaft and by grant GM60904 (J.D.S.) as well as by Cancer Center Support Grant P30 CA21745 (J.D.S.) and the American Lebanese Syrian Associated Charities (J.D.S.).


   Abbreviations
 
ABC, ATP-binding cassette
BD, Becton-Dickinson
LTD4R, leukotriene D4 receptor
Mrp1, multidrug-resistance-associated protein 1

   Notes
 
Transmitting editor: T. Hünig

Received 16 May 2006, accepted 24 August 2006.


   References
Top
 Abstract
 Introduction
 Methods
 Results and Discussion
 References
 

  1. Cole SP, Bhardwaj G, Gerlach JH, et al. (1992) Overexpression of a transporter gene in a multidrug-resistant human lung cancer cell line [see comments]. Science 258:1650.[Abstract/Free Full Text]
  2. Lohoff M, Prechtl S, Sommer F, et al. (1998) A multidrug-resistance protein (MRP)-like transmembrane pump is highly expressed by resting murine T helper (Th) 2, but not Th1 cells, and is induced to equal expression levels in Th1 and Th2 cells after antigenic stimulation in vivo. J. Clin. Invest 101:703.[Web of Science][Medline]
  3. Prechtl S, Roellinghoff M, Scheper R, Cole SP, Deeley RG, Lohoff M. The multidrug resistance protein 1: a functionally important activation marker for murine Th1 cells. J. Immunol. 164:754.
  4. Reiner SL and Locksley RM. (1995) The regulation of immunity to Leishmania major. Annu. Rev. Immunol. 13:151.[CrossRef][Web of Science][Medline]
  5. Zhang J, Alston MA, Huang H, Rabin RL. (2006) Human T cell cytokine responses are dependent on multidrug resistance protein-1. Int. Immunol. 18:485.[Abstract/Free Full Text]
  6. Wijnholds J, Evers R, van Leusden MR, et al. (1997) Increased sensitivity to anticancer drugs and decreased inflammatory response in mice lacking the multidrug resistance-associated protein. Nat. Med. 3:1275.[CrossRef][Web of Science][Medline]
  7. Lohoff M, Ferrick D, Mittrucker HW, et al. (1997) Interferon regulatory factor-1 is required for a T helper 1 immune response in vivo. Immunity 6:681.[CrossRef][Web of Science][Medline]
  8. Lohoff M, Duncan GS, Ferrick D, et al. (2000) Deficiency in the transcription factor interferon regulatory factor (IRF)-2 leads to severely compromised development of natural killer and T helper type 1 cells. J. Exp. Med. 192:325.[Abstract/Free Full Text]
  9. Conseil G, Deeley RG, Cole SP. (2005) Polymorphisms of MRP1 (ABCC1) and related ATP-dependent drug transporters. Pharmacogenet. Genomics. 15:523.[Web of Science][Medline]
  10. Reid G, Wielinga P, Zelcer N, et al. (2003) Characterization of the transport of nucleoside analog drugs by the human multidrug resistance proteins MRP4 and MRP5. Mol. Pharmacol. 63:1094.[Abstract/Free Full Text]
  11. Elliott JI, Raguz S, Higgins CF. (2004) Multidrug transporter activity in lymphocytes. Br. J. Pharmacol. 143:899.[CrossRef][Web of Science][Medline]
  12. Jones TR, Zamboni R, Belley M, et al. (1989) Pharmacology of L-660,711 (MK-571): a novel potent and selective leukotriene D4 receptor antagonist. Can. J. Physiol. Pharmacol. 67:17.[Web of Science][Medline]
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  14. ten Hove T, Drillenburg P, Wijnholds J, Te Velde AA, van Deventer SJ. (2002) Differential susceptibility of multidrug resistance protein-1 deficient mice to DSS and TNBS-induced colitis. Dig. Dis. Sci. 47:2056.[CrossRef][Web of Science][Medline]

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