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Originally published online as doi:10.1189/jlb.0307148 on October 30, 2007

Published online before print October 30, 2007
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(Journal of Leukocyte Biology. 2008;83:368-380.)
© 2008 by Society for Leukocyte Biology

Intracellular zinc homeostasis in leukocyte subsets is regulated by different expression of zinc exporters ZnT-1 to ZnT-9

Silke Overbeck*, Peter Uciechowski*, M. Leigh Ackland{dagger}, Dianne Ford{ddagger} and Lothar Rink*,1

* Institute of Immunology, RWTH Aachen University Hospital, Aachen, Germany;
{dagger} Center for Cellular and Molecular Biology, School of Biological and Chemical Sciences, Deakin University, Victoria, Australia; and
{ddagger} Institute for Cell and Molecular Biosciences, University of Newcastle, The Medical School, Newcastle, United Kingdom

1 Correspondence: Institute of Immunology, RWTH Aachen University Hospital, Pauwelsstrasse 30, D-52074 Aachen, Germany. E-mail: lrink{at}ukaachen.de


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ABSTRACT
 
Intracellular zinc homeostasis is strictly regulated by zinc binding proteins and zinc transporters. In the present study, we quantified in a first global view the expression of all characterized human zinc exporters (hZnT-1-9) in different leukocyte subsets in response to zinc supplementation and depletion and analyzed their influence on alterations in the intracellular zinc concentration. We found that hZnT-1 is the most regulated zinc exporter. Furthermore, we discovered that hZnT-4 is localized in the plasma membrane similar to hZnT-1. hZnT-4 is most highly expressed in Molt-4, up-regulated after treatment with PHA and is responsible for the measured decrease of intracellular zinc content after high zinc exposure. In addition, we found that hZnT-5, hZnT-6, and hZnT-7 in Raji as well as hZnT-6 and hZnT-7 in THP-1 are up-regulated in response to cellular zinc depletion. Those zinc exporters are all localized in the Golgi network, and this type of regulation explains the observed zinc increase in both cell types after up-regulation of their expression during zinc deficiency and, subsequently, high zinc exposure. Furthermore, we detected, for the first time, the expression of hZnT-8 in peripheral blood lymphocytes, which varied strongly between individuals. While hZnT-2 was not detectable, hZnT-3 and hZnT-9 were expressed at low levels. Further on, the amount of expression was higher in primary cells than in cell lines. These data provide insight into the regulation of intracellular zinc homeostasis in cells of the immune system and may explain the variable effects of zinc deficiency on different leukocyte subsets.

Key Words: lymphocytes • immunology • trace elements • cation transport protein • humans


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INTRODUCTION
 
Zinc is an essential trace element, which is a fundamental component of more than 300 metalloenzymes from all six enzyme classes, including transcription factors, and plays an important role in the immune system [1 2 3 ]. Because zinc deficiency results in decreased immune functions [4 ], regulation of intracellular zinc homeostasis by zinc binding proteins and zinc transporters is crucial. While zinc uptake is mediated by members of the SLC39 or Zip (Zrt- and Irt-like proteins) family, zinc export is conducted with ZnT transporter proteins, which belong to the SLC30 or CDF family (cation diffusion facilitator). Fifteen Zip and 9 ZnT transporters have been characterized in human cells [5 ].

ZnT-1 is ubiquitously expressed and the only zinc transporter known to be involved in zinc efflux across the plasma membrane in many different cells, thus conferring resistance to zinc [6 7 8 9 10 11 ]. ZnT-2, which is located in acidic endosomal/lysosomal vesicles, facilitates vesicular zinc accumulation. The tissue distribution for ZnT-2 is primarily the intestine, kidney, placenta, testis, and prostate [12 13 14 ]. It is reported that ZnT-2 is not expressed in the liver, mammary gland, muscle, adipose, thymus, and spleen [7 ]. ZnT-3 is mainly expressed in the membrane of zinc-rich synaptic vesicles within mossy fiber boutons of the hippocampus [15 ] and in testis [16 ]. In neurons ZnT-3 delivers cytoplasmic zinc into synaptic vesicles, which is then released upon excitation [15 ]. ZnT-4 is ubiquitously expressed and located on intracellular vesicles in the kidney and gut, but mostly in the brain and mammary epithelia, where it regulates milk zinc content. A premature translation termination codon in the ZnT-4 gene leads to the phenotype of a lethal milk mouse [17 18 19 20 ]. ZnT-5, which is ubiquitously expressed, but more abundantly so in pancreatic β cells, transports zinc into insulin-containing secretory granules [21 ]. Recently, it was shown that one splice variant of ZnT-5 colocalizes with the plasma membrane in Chinese hamster ovary cells [22 ]. Furthermore, ZnT-5 plays a fundamental role in the maturation of osteoblasts and in the maintenance of normal heart function [23 24 25 26 ]. ZnT-6 mediates zinc transport from cytoplasm into the trans-Golgi apparatus, as well as into the vesicular compartment. While ZnT-6 RNA was detected in the monocytic cell line THP-1, liver, kidney, brain, and intestine, ZnT-6 protein was demonstrated in the brain and lung [10 , 27 ]. ZnT-7 expression is highest in the liver and small intestine with moderate expression in the kidney, spleen, heart, brain, and lung [28 ]. It was also found in THP-1 cells [10 ]. In addition to ZnT-6, ZnT-7 is involved in translocation of the cytoplasmic zinc into the Golgi network [28 ]. ZnT-8 expression is described to be absent in peripheral blood lymphocytes and only restricted to the liver and pancreas, mainly in the islets of Langerhans, where it mediates the relocation of zinc from the cytoplasm into intracellular vesicles, thus providing zinc for insulin maturation and storage [29 , 30 ]. ZnT-9, also known as HUEL, shows an ubiquitous tissue distribution [5 , 31 ].

Recently, it was shown that in dendritic cells, lipopolysaccharide-induced alterations in the expression of zinc transporters altered intracellular zinc concentration and influenced the maturation of the cells [32 ], emphasizing the important role of zinc homeostasis for immune function. Until now, the expression and regulation of zinc transporters and their influence on intracellular zinc homeostasis in cells of the immune system have always been analyzed partially by using different methods. The information that are available so far for the different regulation patterns of each ZnT in response to zinc supplementation, as well as depletion in various cell systems, are listed in Table 1 . In this study, we present the first global view on the expression and distribution of all described human zinc exporter proteins in different leukocyte subsets, including Raji as B cell line, Molt-4 as T cell line and THP-1 as monocytic cell line, as well as in freshly isolated peripheral blood mononuclear cells (PBMCs), primary T and B cells using real-time TaqMan-PCR. Furthermore, we investigated the various regulation patterns due to zinc supplementation and zinc depletion with the membrane permeable chelator N, N, N’, N’-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) in all cell lines, as well as in PBMCs. In addition, we analyzed zinc import and export and determined that intracellular zinc homeostasis is regulated differently, depending on the cell type, which can be attributed to varying expression patterns of zinc exporters in the leukocyte subsets.


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  		Table 1. Tissue Distribution, Subcellular Localization, and Regulation of ZnTs


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MATERIALS AND METHODS
 
Lymphocyte cultures, culture conditions, and isolation of PBMCs, as well as primary T and B cells
All cell lines were obtained from DSMZ GmbH (Braunschweig, Germany). Raji cells and Molt-4 cells were cultured in RPMI 1640 medium (Cambrex, Verviers, Belgium) supplemented with 10% heat-inactivated low-endotoxin fetal calf serum (FCS; PAA Laboratories, Linz, Austria), 100 U/ml penicillin, 100 µg/ml streptomycin and 2 mM L-glutamine (all obtained from Cambrex). In addition, the culture medium for THP-1 cells contained 70 µM β-mercaptoethanol (Merck, Darmstadt, Germany). Caco-2 cells were maintained in DMEM medium (Cambrex) supplemented with 20% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, and 1% nonessential amino acids (Cambrex). PBMCs were isolated from whole blood samples of young healthy donors. After density centrifugation over Ficoll-Hypaque (Biochrom, Berlin, Germany), the cells of the interface were collected, washed twice with PBS (Cambrex), and resuspended in RPMI1640 medium. Primary human T and B cells were isolated using the Dynal T Cell Negative Isolation Kit ver. II and Dynal B Cell Negative Isolation Kit (Invitrogen, Karlsruhe, Germany), respectively, according to the manufacturer’s instructions and resuspended in RPMI1640 medium. All cells were grown, and all incubation steps were carried out at 37°C and 5% CO2 in a humidified atmosphere.

Reagents
Zinc sulfate (Sigma-Aldrich, Steinheim, Germany) was dissolved in sterile water to obtain a stock solution of 100 mM, which was then sterile filtered. This stock was further diluted in nonsupplemented protein-free medium (Ultradoma P.F., Cambrex) in order to achieve a final concentration of 2 mM, which was used for experiments. TPEN (Sigma-Aldrich) was dissolved in sterile water to obtain a stock solution of 2 mM, which was ready to use after sterile filtration. Phytohemagglutinin (PHA; Becton Dickinson, Heidelberg, Germany) was dissolved in RPMI 1640 medium supplemented with 10% FCS to obtain a stock solution of 1 mg/ml, which was then sterile filtered.

Relative quantification of hZnT transporters
Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Isolated mRNA was transcribed into cDNA with the Reverse Transcription System (Promega, Madison, WI, USA) and was performed according to the manufacturer’s instructions. For quantification of the mRNA levels of hZnT-1-9, a sequence detection system (7000, Applied Biosystems, Foster City, CA, USA) was used. The oligonucleotide primers, which are located in adjacent exons so one can exclude amplification of genomic DNA, and the FAM/TAMRA-labeled TaqMan probes were designed with Primer Express software (Applied Biosystems) (Table 2 ). PBGD (porphobilinogen deaminase) was chosen (accession no. NM_000190) as the housekeeping gene, since GAPDH (glyceraldehyde-3-phosphate dehydrogenase), which is a common housekeeping gene, displayed a regulation due to stimulation conditions. The sequences of these primers and the probe have previously been reported [33 ]. For quantification, the comparative CT method ({Delta}{Delta}CT [cycle threshold]) [34 , 35 ] was used, in which the resulting mRNA levels of the hZnTs samples were normalized to the housekeeping gene mRNA levels as an endogenous reference. The single results were then put into an equation referring to a standard curve, which was initially established for each zinc transporter. This allows us to compare the results from one set of cells to another one. The components of each PCR (50 µl) were 30 µl dH2O, 5 µl PCR-buffer (Qiagen), 5 µl of magnesium chloride (25 mM; Qiagen), 1 µl dNTPs (10 mM; Applied Biosystems), 0.75 µl forward and reverse primer each (20 µM; Tib Molbiol, Berlin, Germany), 0.5 µl ROX (100 mM; Tib Molbiol), 0.5 µl probe (20 µM; Tib Molbiol), 0.5 µl HotStar-Taq (5 U/µl, Qiagen), and 6 µl of the respective cDNA or dH2O as a negative control. Amplification was done up to 45 cycles. After HotStar-Taq activation (15 min at 95°C), each cycle consisted of 2 steps, denaturing (15 s at 95°C) and annealing/extending (60 s at 60°C). All assays were run in triplicate.


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  		Table 2. TaqMan Primers and Probes for Quantification of hZnT Gene Expression

Detection of hZnT-1 protein using Western blot analysis
Cells were lysed in a buffer containing 65 mM Tris-HCl (pH 6.8), 25% glycerin, 2% sodium dodecyl sulfate, and 1 mM sodium orthovanadate. Samples were sonicated twice for 10 s (Vibracell, Sonics and Materials, Danbury, CT, USA) and then incubated for 5 min at 95°C. After determining the protein concentration with the Bio-Rad protein assay (Bio-Rad, Munich, Germany), 30 µg of total protein was subjected to sodium dodecyl sulfate-PAGE (SDS-PAGE) on an 8.5% acrylamide gel. Proteins were transferred onto nylon membranes (Bio-Rad), which were stained with Ponceau S solution (Sigma-Aldrich, Steinheim, Germany) to determine equal protein loading. Membranes were then blocked overnight in a buffer containing 20 mM Tris-base, 140 mM sodium chloride, 0.1% Tween-20, and 5% nonfat milk powder (pH 7.6) followed by incubation with affinity purified anti-ZnT-1 (1:100 dilution) [36 ] for 3 h at 4°C in the same buffer mentioned above. After three washing steps, the nylon membranes were incubated for 1 h at room temperature with a horseradish peroxidase conjugated goat anti-rabbit IgG secondary antibody (1:2000 dilution) (Cell Signaling, Danvers, MA, USA). The resulting peroxidase activity was visualized using LumiGLO (Cell Signaling) according to the manufacturer’s instructions.

Detection of hZnT-4 protein on cell surface using flow cytometry and fluorescence microscopy
Adherent cells, grown in a monolayer, or 1x106 cells in suspension, respectively, were incubated with 10% AB serum at 37°C for 10 min. After washing the cells with PBS, another incubation step with 4% paraformaldehyde in HBSS (Hank's balanced salt solution) at 4°C for 20 min was performed. Afterward, the cells were washed twice with PBS before a 0.1% saponin in HBSS solution containing a rabbit anti-human ZnT-4 antibody (1:20 dilution) [19 ] was added for 20 min at 4°C. Subsequently, unbound antibodies were removed by two washing steps with 0.1% saponin in HBSS solution. Then, the cells were incubated with a FITC-labeled goat anti-rabbit IgG secondary antibody (1:100 dilution) (Dianova, Hamburg, Germany) in 0.1% saponin in HBSS solution for another 20 min at 4°C. After washing the cells with PBS, the fluorescence was detected using a flow cytometer (FACS-Calibur, Becton Dickinson) and a fluorescence microscope (Axioskop, Zeiss, Jena, Germany).

Analysis of intracellular zinc concentration
One million cells per ml were loaded with 1 µM Fluozin-3 AM ester (Invitrogen, Karlsruhe, Germany) in a buffer containing 5 mM glucose (Merck), 1 mM magnesium chloride (Merck), 1 mM sodium dihydrogen phosphate (Sigma-Aldrich), 1.3 mM calcium chloride (Merck), 25 mM HEPES (Merck), 120 mM sodium chloride (Merck), 5.4 mM potassium chloride (Merck), and 10% FCS (pH 7.35). Incubation was carried out at 37°C for 30 min in a shaking water bath. After pelleting the cells, the density was adjusted to 2 x 106 cells/ml. One hundred microliters of cells per well were then distributed into a 96-well plate and partially stimulated with 50 µM TPEN or 100 µM zinc and 50 µM sodium pyrithione (Sigma-Aldrich) to obtain minimal and maximal zinc concentration, respectively. After incubating the cells at 37°C for 15 min., the remaining cells were stimulated with 50 µM zinc. The resulting fluorescence was measured over a period of two and a half hours with a TECAN 340 fluorescence multiwell plate reader (Tecan, Crailsheim, Germany) using excitation and emission wavelengths of 485 nm and 535 nm, respectively. The concentration of intracellular labile zinc was calculated with the formula: [Zn] = KD x [(F–Fmin)/(Fmax–F)] [37 ]. The dissociation constant of the Fluozin-3/zinc complex is 15 nM [38 ].

Statistical analysis
The statistical analyses were performed with Student's t test using the software SigmaPlot (SPSS, Chicago, IL, USA).


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RESULTS
 
Differential hZnT expression in leukocyte subsets
Using TaqMan-PCR, we quantified the expression of all known human zinc exporters, hZnT-1 to hZnT-9, in Raji (B cell line), Molt-4 (T cell line), THP-1 (monocytic cell line) (Fig. 1A ), and freshly isolated PBMCs (Fig. 1B) . The leukocyte subsets differed strongly in their distribution and their amount of expression of the single transporters. Expression of hZnT-1 reached a level in PBMCs, which was 40 times higher than in Raji, 60 times higher than in THP-1, and even 390 times higher than in Molt-4. No expression of hZnT-2 could be detected either in cell lines or in PBMCs. hZnT-3 was found to a low degree in THP-1 and PBMCs and to a nearly undetectable level in Molt-4. PBMCs and Molt-4 showed distinct hZnT-4 mRNA expression, while the level was low in Raji and THP-1. mRNA of hZnT-5, hZnT-6, and hZnT-7 was detected in all analyzed cells, with the highest expression in PBMCs followed by THP-1, Molt-4, and then Raji. hZnT-8 was found in PBMCs and to a lesser degree in Molt-4. Interestingly, hZnT-8 mRNA was not constitutively present in each PBMC (Fig. 1C) , which was only seen for this zinc transporter. hZnT-8 expression varied greatly among individuals, ranging from PBMCs being negative to strongly positive for hZnT-8 mRNA. The expression of hZnT-9 was at a similar low level in all investigated cells.


Figure 1
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  		Figure 1. hZnT gene expression in Molt-4, Raji, THP-1, and PBMCs. T cells (Molt-4), B cells (Raji), monocytes (THP-1), and PBMCs were cultured for 40 h. Relative mRNA quantification was carried out by real-time TaqMan-PCR. hZnT mRNA was normalized to PBGD mRNA. (A) Comparison of selective mRNA expression in Molt-4, Raji and THP-1. (B) hZnT mRNA expression in PBMCs. (C) Individual hZnT-8 mRNA expression in 7 healthy donors. (D) Agarose gel electrophoresis of hZnT-1 through hZnT-9 in all investigated cell types. (E) Agarose gel electrophoresis of hZnT-2 in prostate cDNA, which served as positive control. Mean values ± SE of n = 3 (A and B, n=7 for hZnT-8) or one representative experiment out of three is shown (C–E). L, Ladder; 1, Raji; 2, Molt-4; 3, THP-1; 4, PBMC; 5, negative control; and p, prostate positive control.

The size of the resultant PCR products and PCR specificity were verified with agarose gel electrophoresis. Following TaqMan-PCR, hZnTs of the predicted size were detected (Fig. 1D) . Because hZnT-2 could not be detected, prostate cDNA (kindly provided by Prof. Schulz, Düsseldorf, Germany) served as a positive control, thus confirming the functionality of the selected primer pair (Fig. 1E) .

Altogether, under physiological conditions hZnT-1 was the main transporter in Raji and PBMCs, whereas hZnT-5 and hZnT-6 were most highly expressed in THP-1 and Molt-4, respectively (Fig. 1A and 1B) .

To determine whether the used cell lines are comparable with primary human monocytes, T and B cells, we analyzed the expression of hZnTs in isolated cells. Concerning the T cells, which represent ~60% of PBMCs, the isolation was successful and reached a purity of 97.3% on average (unpublished results). With regard to the B cells, which represent ~20% of PBMCs, a purity of 95.6% on average was achieved (unpublished results), but only a low yield of B cells and correspondingly mRNA could be isolated, which made it necessary to pool the mRNA of three different donors before analysis in order to run one PCR reaction. Isolation of enough monocyte mRNA could not be performed.

The expression profile of hZnT-1 to hZnT-9 in primary human T cells was similar to that in the T cell line Molt-4, even though the values for each single transporter were much higher in primary T cells (Fig. 2A ). There was no expression of hZnT-2 and a nearly undetectable level of hZnT-3 expression, while a distinct expression of hZnT-4 could be measured. Again, hZnT-6 was the main transporter. Furthermore, there was a high expression of hZnT-7, while the expression levels of hZnT-8 and hZnT-9 were low. Surprisingly, there was a distinct expression of hZnT-1, which could not be observed in the T cell line, and the expression of hZnT-5 was not as high as in the T cell line when compared with the other zinc transporters.


Figure 2
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  		Figure 2. hZnT gene expression in primary T and B cells. RNA of primary T and B cells was isolated directly after purification. Relative mRNA quantification was carried out by real-time TaqMan-PCR. hZnT mRNA was normalized to PBGD mRNA. (A) hZnT mRNA expression in primary T cells. Mean values ± SE of n = 3. (B) hZnT mRNA expression in primary B cells. Expression value of hZnT mRNA pooled from n = 3 different blood donors.

The expression profile of hZnT-1 to hZnT-9 in primary human B cells displayed similar high values for each single transporter like in primary T cells, but some more differences could be observed when compared with the B cell line Raji (Fig. 2B) . In this case, although there was a distinct expression of hZnT-1, now hZnT-6 was the main transporter. In contrast to the B cell line, the expression of hZnT-3 and hZnT-8 could be detected; there was a strong expression of hZnT-4 and only a low expression of hZnT-5, while the expression of hZnT-7 was higher when compared with the other zinc transporters. As measured in the B cell line, there was no expression of hZnT-2 and a low expression of hZnT-9.

Because of the overall higher expression of zinc transporters in primary cells, in comparison to the proliferating cell lines, we analyzed the expression of hZnT-1 to hZnT-9 in primary unstimulated T cells and T cells, which were stimulated with PHA (10 µg/ml) for 48 h in order to induce proliferation. Both expression profiles resembled each other, but the extent of expression was clearly decreased in stimulated T cells (Fig. 3 ).


Figure 3
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  		Figure 3. hZnT gene expression in activated primary T cells. Primary T cells were stimulated with 10 µg/ml PHA for 48 h. Relative mRNA quantification was carried out by real-time TaqMan-PCR. hZnT mRNA was normalized to PBGD mRNA. hZnT mRNA expression in primary T cells before and after stimulation with PHA. One representative experiment is shown.

Regulation of hZnT expression
To investigate the influence of zinc supplementation and zinc depletion on hZnT expression, the different cell lines and PBMCs were cultured for 40 h with 15 µM or 30 µM zinc, respectively, or 2.5 µM TPEN. PBMCs and Molt-4 were stimulated with 1 µM TPEN, because cells were not viable after incubation with higher concentrations, as detected by propidium iodide staining (unpublished results).

Expression of hZnT-1, whose mRNA was regulated most clearly among all investigated zinc transporters, rose with increasing zinc concentrations in all cell lines, as well as in PBMCs (Fig. 4 ). Zinc concentrations ranging from 15 to 30 µM affected the cells differentially. In Molt-4 and THP-1, hZnT-1 mRNA was significantly up-regulated 12-fold and 20-fold in response to 15 µM or 30 µM zinc (Fig. 4A and 4B) , respectively, while the expression in Raji and PBMCs was up-regulated only twofold and threefold, respectively (Fig. 4C and 4D) . Under zinc deprivation with TPEN, mRNA expression was slightly down-regulated in comparison to the unstimulated sample, which could be most clearly seen in THP-1 (Fig. 4) . The results obtained for mRNA analysis could also be confirmed on the protein level in all investigated leukocyte subsets (Fig. 4) .


Figure 4
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  		Figure 4. hZnT-1 expression in Molt-4, THP-1, Raji, and PBMCs. Cells were stimulated with 15 µM zinc, 30 µM zinc, 2.5 µM TPEN (THP-1 and Raji), and 1 µM TPEN (Molt-4 and PBMCs) for 40 h. Relative mRNA quantification was carried out by real-time TaqMan-PCR. hZnT mRNA was normalized to PBGD mRNA. (A) hZnT-1 mRNA and protein expression in Molt-4. (B) hZnT-1 mRNA and protein expression in THP-1. (C) hZnT-1 mRNA and protein expression in Raji. (D) hZnT-1 mRNA and protein expression in PBMCs. Under the graphs, a corresponding Western blot for hZnT-1 is shown. Mean values ± SE of n = 3 (TaqMan PCR) or one representative experiment out of three (Western blot) is shown. *, P < 0.05, **, P < 0.01 and ***, P < 0.001. C, control; TP, TPEN.

mRNA levels of hZnT-3 were slightly altered in Molt-4 and PBMCs in response to zinc excess, as well as zinc depletion (Fig. 5A and 5B ), but one has to consider that this happened overall on a low expression level. In THP-1, zinc supplementation did not change the hZnT-3 level, while zinc deprivation resulted in a down-regulation of mRNA expression, even at low levels (P=0.06) (Fig. 5C) .


Figure 5
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  		Figure 5. hZnT-3 expression in Molt-4, PBMCs, and THP-1. Cells were stimulated with 15 µM zinc, 30 µM zinc, 2.5 µM TPEN (THP-1 and Raji) and 1 µM TPEN (Molt-4 and PBMCs) for 40 h. Relative mRNA quantification was carried out by real-time TaqMan-PCR. hZnT mRNA was normalized to PBGD mRNA. Because of the lack of hZnT-3 mRNA expression in B cells, these data are not shown. (A) hZnT-3 mRNA expression in Molt-4. (B) hZnT-3 mRNA expression in PBMCs. (C) hZnT-3 mRNA expression in THP-1. Mean values ± SE of n = 3. C, control, TP, TPEN.

Expression of hZnT-4 mRNA was not altered with zinc supplementation or following zinc depletion in Molt-4 and PBMCs (Fig. 6A and 6B ). In Raji and THP-1, a slight regulation in response to 15 µM zinc and 2.5 µM TPEN was detected, but this occurred at a low level (Fig. 6C and 6D) . Using flow cytometry and fluorescence microscopy, we detected expression of hZnT-4 at the protein level, which confirms the mRNA analysis (Fig. 7A and 7C ). We found that the mean fluorescence slightly decreased after TPEN treatment (91%) in comparison to the control. Interestingly, we were able to show for the first time that this transporter is localized in the plasma membrane of Molt-4 (Fig. 7C) , Raji, THP-1, and PBMCs (unpublished results), which is, until now, only described for ZnT-1 in many different cells [5 ] and ZnT-5 in Chinese hamster ovary cells [22 ]. Caco-2 cells, derived from a human colon adenocarcinoma, were also stained with anti-ZnT-4, showing clearly detectable intracellular staining (Fig. 7B) , similar to that described for PMC42 cells by Michalczyk et al. [19 ]. This staining differed strongly in comparison to the plasma membrane localization in cells of the immune system. Another interesting point is the significant up-regulation of hZnT-4 in PBMCs after stimulation with the mitogen PHA (10 µg/ml) for 40 h (Fig. 7D) , which indicates a role for hZnT-4 in the activation of leukocytes during immune responses. This seems to be in contradiction with the low expression of hZnT-4 in PHA stimulated primary T cells after 48 h, but using isolated primary T cells we could demonstrate that the mRNA expression of hZnT-4 is already induced one hour after stimulation with PHA (10 µg/ml) (Fig. 7E) . Subsequently, the expression level of hZnT-4 decreases (unpublished results).


Figure 6
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  		Figure 6. hZnT-4 expression in Molt-4, PBMCs, Raji, and THP-1. Cells were stimulated with 15 µM zinc, 30 µM zinc, 2.5 µM TPEN (THP-1 and Raji), and 1 µM TPEN (Molt-4 and PBMCs) for 40 h. Relative mRNA quantification was carried out by real-time TaqMan-PCR. hZnT mRNA was normalized to PBGD mRNA. (A) hZnT-4 mRNA expression in Molt-4. (B) hZnT-4 mRNA expression in PBMCs. (C) hZnT-4 mRNA expression in Raji. (D) hZnT-4 mRNA expression in THP-1. Mean values ± SE of n = 3. C, control; TP, TPEN.


Figure 7
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  		Figure 7. hZnT-4 protein localization in Molt-4 and Caco-2. Molt-4 cells were stimulated with 15 µM zinc, 30 µM zinc or 1 µM TPEN. PBNCs were stimulated with 10 µg/ml PHA for 40 h. Caco-2 cells were cultured for 40 h. hZnT-4 protein was detected by flow cytometry and fluorescence microscopy. Primary T cells were stimulated with 10 µg/ml PHA for 1 h. Relative mRNA quantification was carried out by real-time TaqMan-PCR. hZnT mRNA was normalized to PBGD mRNA. (A) hZnT-4 protein expression in Molt-4. (B) Localization of hZnT-4 protein in Caco-2 cells. (C) Localization of hZnT-4 protein in Molt-4. (D) hZnT-4 protein expression in PBMCs after stimulation with PHA. (E) hZnT-4 mRNA expression in primary T cells after stimulation with PHA. One representative experiment out of three is shown (A–C, E) or mean values ± SE of n = 3 (D). **, P ≤ 0.01.

The mRNA level of hZnT-5 did not change during zinc treatment and zinc deprivation in Molt-4 (Fig. 8A ). Surprisingly, in Raji an up-regulation of hZnT-5 during zinc deficiency occurred (Fig. 8B) , which could not be observed in THP-1 and PBMCs under the same conditions (Fig. 8C and 8D) . Under zinc supplementation, hZnT-5 expression decreased slightly in PBMCs and significantly in THP-1 (Fig. 8C and 8D) .


Figure 8
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  		Figure 8. hZnT-5 expression in Molt-4, Raji, THP-1, and PBMCs. Cells were stimulated with 15 µM zinc, 30 µM zinc, 2.5 µM TPEN (THP-1 and Raji), and 1 µM TPEN (Molt-4 and PBMCs) for 40 h. Relative mRNA quantification was carried out by real-time TaqMan-PCR. hZnT mRNA was normalized to PBGD mRNA. (A) hZnT-5 mRNA expression in Molt4. (B) hZnT-5 mRNA expression in Raji. (C) hZnT-5 mRNA expression in THP-1. (D) hZnT-5 mRNA expression in PBMCs. Mean values ± SE of n = 3. *, P < 0.05. C, control; TP, TPEN.

hZnT-6 and hZnT-7 showed both the same regulation pattern. In Raji and THP-1, expression did not change in response to different zinc concentrations, while the mRNA was significantly up-regulated after zinc deprivation comparable to hZnT-5 in Raji (Fig. 9A and 9B ; Fig. 10A and 10B ). In Molt-4 and PBMCs, expression of both zinc transporters was unaffected in response to zinc supplementation, as well as zinc depletion (Fig. 9C and 9D and Fig. 10C and 10D ).


Figure 9
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  		Figure 9. hZnT-6 expression in Molt-4, Raji, THP-1, and PBMCs. Cells were stimulated with 15 µM zinc, 30 µM zinc, 2.5 µM TPEN (THP-1 and Raji), and 1 µM TPEN (Molt-4 and PBMCs) for 40 h. Relative mRNA quantification was carried out by real-time TaqMan-PCR. hZnT mRNA was normalized to PBGD mRNA. (A) hZnT-6 mRNA expression in Raji. (B) hZnT-6 mRNA expression in THP-1. (C) hZnT-6 mRNA expression in Molt-4. (D) hZnT-6 mRNA expression in PBMCs. Mean values ± SE of n = 3. *, P < 0.05. C, control; TP, TPEN.


Figure 10
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  		Figure 10. hZnT-7 expression in Molt-4, Raji, THP-1, and PBMCs. Cells were stimulated with 15 µM zinc, 30 µM zinc, 2.5 µM TPEN (THP-1 and Raji), and 1 µM TPEN (Molt-4 and PBMCs) for 40 h. Relative mRNA quantification was carried out by real-time TaqMan-PCR. hZnT mRNA was normalized to PBGD mRNA. (A) hZnT-7 mRNA expression in Raji. (B) hZnT-7 mRNA expression in THP-1. (C) hZnT-7 mRNA expression in Molt-4. (D) hZnT-7 mRNA expression in PBMCs. Mean values ± SE of n = 3. *, P < 0.05. C, control; TP, TPEN.

PBMCs, which were positive for hZnT-8 mRNA expression (Fig. 1C) , showed no regulation (Fig. 11A ). Neither zinc supplementation nor zinc deficiency changed the mRNA level. In contrast, hZnT-8 expression in Molt-4 was reduced after TPEN treatment, even if the expression was overall low (Fig. 11B) . Zinc excess had no effect on the hZnT-8 mRNA level in Molt-4.


Figure 11
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  		Figure 11. hZnT-8 expression in PBMCs and T cells. Cells were stimulated with 15 µM zinc, 30 µM zinc, 2.5 µM TPEN (THP-1 and Raji), and 1 µM TPEN (Molt-4 and PBMCs) for 40 h. Relative mRNA quantification was carried out by real-time TaqMan-PCR. hZnT mRNA was normalized to PBGD mRNA. Because of the lack of hZnT-8 mRNA expression in Raji and THP-1, these data are not shown. (A) hZnT-8 mRNA expression in PBMCs. (B) hZnT-8 mRNA expression in Molt-4. Mean values ± SE of n = 7 for PBMCs and n =3 for T cells. C, control; TP, TPEN.

Different zinc concentrations and zinc depletion did not influence the expression of hZnT-9 in Molt-4, THP-1, and PBMCs (Fig. 12A 12B 12C ). Only in Raji a slight up-regulation of hZnT-9 in response to zinc deficiency could be observed (Fig. 12D) .


Figure 12
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  		Figure 12. hZnT-9 expression in Molt-4, Raji, THP-1, and PBMCs. Cells were stimulated with 15 µM zinc, 30 µM zinc, 2.5 µM TPEN (THP-1 and Raji) and 1 µM TPEN (Molt-4 and PBMCs) for 40 h. Relative mRNA quantification was carried out by real-time TaqMan-PCR. hZnT mRNA was normalized to PBGD mRNA. (A) hZnT-9 mRNA expression in Molt-4. (B) hZnT-9 mRNA expression in THP-1. (C) hZnT-9 mRNA expression in PBMCs. (D) hZnT-9 mRNA expression in Raji. Mean values ± SE of n = 3. C, control; TP, TPEN.

Altogether, after zinc supplementation, hZnT-1 was the main transporter in all investigated cell types.

Regulation of intracellular zinc homeostasis
To analyze how the unexpected up-regulation of zinc exporters induced by zinc depletion influenced intracellular zinc homeostasis, the different cell lines were cultured for 40 h with 1 µM (Molt-4) or 2.5 µM TPEN (Raji and THP-1). Subsequently, the cells were stimulated with 50 µM zinc and zinc import and export were measured with Fluozin-3 over a period of two and a half hours.

At time point zero, the labile zinc concentration in Molt-4 was 0.11 nM in the control and 0.08 nM in the TPEN-stimulated sample (Fig. 13A ). After a stimulation period of 16 min with 50 µM zinc, Molt-4 cells reached the highest intracellular zinc concentration (44 nM), which was independent from the prestimulation with TPEN. After this, the zinc concentration in Molt-4 was reduced as a result of zinc efflux. This effect was greatest in the control (25.3 nM±1.9) followed by the TPEN-stimulated sample (36.8 nM±4.6). At time point zero, the intracellular zinc concentration in Raji was 0.12 nM in the control and 0.08 nM in the TPEN-stimulated sample (Fig. 13B) . After a stimulation period of 16 min. with 50 µM zinc, the labile zinc in Raji increased to 27 nM, which was also independent from the prestimulation with TPEN. While this intracellular zinc concentration was maintained in the unstimulated sample after 148 min., the labile zinc content increased significantly in the TPEN-stimulated sample to 62.7 nM (±4.4). THP-1 cells showed a comparable zinc kinetic to Raji. At time point zero, the labile zinc concentration in THP-1 was 0.24 nM in the control and 0.17 nM in the TPEN-stimulated sample (Fig. 13C) . After a stimulation period of 16 min with 50 µM zinc, the intracellular zinc concentration in unstimulated and with TPEN-stimulated THP-1 cells was 36 nM. After 148 min, the zinc concentration was the same in the control but increased significantly in the TPEN-stimulated sample to 54.5 nM (±0.6).


Figure 13
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  		Figure 13. Regulation of intracellular zinc homeostasis in Molt-4, Raji, and THP-1. Cells were prestimulated with 2.5 µM TPEN (THP-1 and Raji) or 1 µM TPEN (Molt-4) for 40 h. Subsequently, cells were stimulated with 50 µM zinc. Zinc import and export were measured using Fluozin-3. Mean values ± SE of n = 3. **, P ≤ 0.01.


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DISCUSSION
 
Because of the essential role of zinc in the immune system [1 2 3 ], it is crucial to understand, which molecules are important for zinc transport into and out of the cell under different zinc concentrations. This will provide important information about how zinc functions to regulate the activity of the zinc transporters. So far, the expression and regulation of zinc transporters and their influence on intracellular zinc homeostasis in immunologically relevant cells have always been analyzed partially by using different methods. In this study, we present the first global view on the distribution and regulation of all described human zinc exporter proteins in different leukocyte subsets.

Among all transporters, hZnT-1 was the most highly regulated, where expression levels increased up to 20-fold during zinc supplementation. Furthermore, after zinc excess hZnT-1 was the main transporter in all investigated leukocyte subsets and PBMCs. This result emphasizes the important role of hZnT-1 in immune cells and suggests an important contribution of this transporter to immune function. The mRNA results are compatible with the data obtained on the protein levels, thus validating the results. Our findings are consistent with other observations by which zinc supplementation induces ZnT-1 mRNA expression in THP-1 cells, while zinc depletion results in ZnT-1 mRNA reduction [9 , 10 ]. Furthermore, the ZnT-1 mRNA level is described to increase in the small intestine, liver, and kidney of rats after dietary zinc intake [7 ]. In addition, this kind of zinc responsiveness was seen with freshly isolated human leukocytes as well, although this effect disappeared after the cells were used under culture conditions [39 ]. This overall pronounced regulation could be due to the fact that ZnT-1 is, so far, the only described zinc transporter, which is localized in the plasma membrane of many different cells, where it lowers intracellular zinc content by exporting zinc out of the cell [6 ]. In this way, ZnT-1 may confer resistance to zinc and protects the cell from toxicity, as described for transfected baby hamster kidney cells [6 , 8 ]. Regulation of mouse ZnT-1 resembles that of the metallothionein gene and is based on two metal response elements in the ZnT-1 promoter region, which are activated by binding through a six zinc-finger transcription factor, known as the metal response element binding transcription factor-1 [40 ].

Recently, it was shown that, in addition to the plasma membrane expressed ZnT-1, one splice variant of ZnT-5 colocalizes with the plasma membrane in Chinese hamster ovary cells [22 ]. In this study, we were able to identify another potential zinc exporter to be localized in the plasma membrane. Expression of hZnT-4 could be detected in PBMCs, Molt-4 and to a lesser degree in Raji and THP-1 cells. Until now, the localization of ZnT-4 is described to be only present in intracellular vesicles, as has been reported for Caco-2 cells [41 ], HC11 cells [11 ], as well as for the human breast carcinoma cell line PMC42, which showed a granular distribution [19 ], but we were able to demonstrate for the first time that hZnT-4 is also located in the plasma membrane. This staining pattern was restricted to cells of the immune system, because colon-derived Caco-2 cells were stained throughout the whole cytoplasm, excluding the nucleus. It seems, that in contrast to the ubiquitous plasma membrane expression of ZnT-1, different zinc exporter proteins are specifically expressed at the plasma membrane in various cells. In addition, the expression of hZnT-4 was up-regulated already 1 h after stimulation with PHA, which corresponds to an altered physiological state that can be observed during immune responses. This indicates that hZnT-4 participates in the regulation of intracellular zinc concentration during the activation process of leukocytes. Furthermore, it seems that because of the essential role of zinc for the immune system and thereby associated the optimal functionality of each cell type [1 2 3 ], it is important to have two zinc exporter molecules located in the plasma membrane for maintaining a well-balanced intracellular zinc homeostasis.

Under physiological conditions hZnT-5 was the main transporter in THP-1 confirming data obtained by Cousins et al. [9 ]. Surprisingly, expression of hZnT-5 was slightly down-regulated after zinc supplementation in THP-1 and PBMCs. Previous studies indicate that zinc excess results in increased ZnT-5 expression in Caco-2 cells, while no changes could be seen in placental cells and HeLa cells [24 25 26 ]. Furthermore, a down-regulation of the hZnT-5 mRNA level was observed after zinc treatment with 200 µM zinc in Caco-2 cells and a down-regulation of the corresponding protein could be seen in patients, who have had a daily zinc supplement of 25 mg zinc for 14 days [36 ]. These data indicate that hZnT-5 mRNA expression and its regulation varies between different cell types and is dependent on extracellular zinc levels. Another striking effect was the observed up-regulation of hZnT-5 expression after zinc deprivation in Raji and furthermore, the up-regulation of hZnT-6 and hZnT-7 expression during zinc depletion in Raji and THP-1. A comparable mode of action was also seen in TPEN-treated HeLa cells for the expression of ZnT-5 and ZnT-7 [25 ]. This common responsiveness to zinc deficiency could be due to the fact that these three zinc transporters, which are all located in the secretory apparatus, are required for the activation of zinc-containing enzymes by forming homo- and hetero-oligomers and thus transporting zinc into the lumen of the Golgi apparatus and the vesicular compartments [42 , 43 ]. This hypothesis can further be confirmed by the observed increase in intracellular labile zinc concentration in Raji and THP-1. Under zinc deficiency, Raji cells showed the highest zinc uptake, which can be explained by a zinc transport into the secretory apparatus as a consequence of the up-regulation of three zinc exporters (hZnT-5, hZnT-6, and hZnT-7) followed by THP-1 cells, which displayed a higher mRNA expression of two zinc exporters, namely hZnT-6 and hZnT-7, after zinc depletion. Both cells may thus lower their cytoplasmic intracellular zinc levels and maintain their protein synthesis by transporting zinc into the Golgi network. In contrast to this regulation pattern, Molt-4 cells showed a decrease in intracellular labile zinc, which can be explained by the zinc export activity of the newly discovered membrane-localized hZnT-4, which is highest expressed in Molt-4, and the lacking up-regulation of hZnT-5, hZnT-6, and hZnT-7. The slower decrease of the intracellular zinc concentration in the TPEN-stimulated sample in comparison to the unstimulated one can be explained by a lower protein expression of hZnT-4, which was measured using the mean fluorescence intensity. This kind of regulation is more pronounced in the B cell and monocytic cell line, which is in accordance with their higher capacity for protein synthesis when compared with T cells. hZnT-4 seems to work very efficiently, because the Molt-4 was, despite their low-level expression of hZnT-1, the only leukocyte subset, which lower their intracellular zinc content by transporting the zinc outside the cell. Furthermore, the combined activity of hZnT-5, hZnT-6, and hZnT-7, which could also be demonstrated by other research groups [42 , 43 ], seems to be more efficient in comparison to hZnT-1.

Expression of hZnT-2 could not be detected in any of the analyzed cells. Functionality of the selected primer pair was proven by mRNA detection in the prostate. This result confirms earlier data, which exclude mRNA expression in immunologic organs such as the thymus and spleen [9 , 16 ]. Possibly despite zinc, there are other factors that regulate hZnT-2 mRNA expression. This was shown for the zinc importer Zip14, which is up-regulated by IL-6 in the liver and thus may account for the hypozincemia during the acute-phase response [44 ].

Expression of hZnT-3 mRNA was overall low, which verifies the data obtained by Andree et al. [45 ], who did not detect mRNA expression of hZnT-3 in a human lymphoblastoid cell line, which was transformed by Epstein-Barr virus. This further supports a specific role for ZnT-3 in brain and testis [15 , 16 ].

To date, expression of hZnT-8 mRNA has only been detected in the liver and pancreas, mainly in the islets of Langerhans, and not in PBMCs [29 , 30 ]. In this study, we were able to demonstrate hZnT-8 expression to a lesser degree in Molt-4 and, for the first time, to a higher degree in PBMCs, where the expression varies strongly among individuals. These large differences could be the reason why other authors did not see any expression. Zinc excess, as well as zinc deficiency, did not alter the expression. In Molt-4, the hZnT-8 mRNA level decreased during zinc depletion, while zinc supplementation did not change the expression. Future investigations will focus on whether there exists a linkage between hZnT-8 expression and diabetes, because until now, expression is described to be restricted to the liver and pancreas. Regarding the pancreas, hZnT-8 expression is mainly localized in the islets of Langerhans, where it mediates the relocation of zinc from the cytoplasm into intracellular vesicles, thus providing zinc for insulin maturation and storage [29 , 30 ], which resembles the function of hZnT-5 transporting zinc into insulin-containing secretory granules [21 ].

Low-level expression of hZnT-9 could be detected in all analyzed cells. Our findings are consistent with other observations, which showed qualitatively an expression of hZnT-9 (HUEL) in Molt-4, Raji, and peripheral blood leukocytes [31 ]. We could further detect hZnT-9 mRNA in THP-1 cells. The expression was only slightly up-regulated in Raji after TPEN treatment. These data indicate that hZnT-9 plays no important role in the immune system.

The first available comparison of the expression profile of hZnT-1 to hZnT-9 in cell lines with that in primary cells revealed that the amount of expression for each single transporter is higher in primary cells when compared with cell lines. This overall stronger expression could be down-regulated after activating primary T cells with PHA, which induces proliferation. Together with the partially altered expression profile, it seems that in order to maintain intracellular zinc homeostasis, resting and proliferating cells display differences in the expression of zinc exporter proteins, which depend on their different physiological state.

Although hZnT-1, hZnT-4, hZnT-5, hZnT-6, and hZnT-7 are fundamental for maintaining zinc homeostasis and thereby associated the optimal functionality of leukocytes, hZnT-9, as well as hZnT-3 and hZnT-2, seem to play a minor role in the immune system. Another zinc exporter, hZnT-8, will be of future interest with regard to elucidating its role since it varies interindividually like no other transporter. Lastly, hZnT-4 with its membrane expression and its up-regulation during the activation process seems to be a new important regulator for zinc homeostasis exclusively for leukocytes. In conclusion, our data provide insight into the regulation of intracellular zinc homeostasis in cells of the immune system and may explain the different effects on leukocyte subsets observed during zinc deficiency.


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ACKNOWLEDGEMENTS
 
This study was supported in part by the EU project ZINCAGE (Food-CT-2003-506850). We thank Ms. Romney Haylett for critical reading of the manuscript.

Received March 12, 2007; revised September 21, 2007; accepted October 6, 2007.


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