Zinc transporter ZIP8 (SLC39A8) and zinc influence IFN- expression in activated human T cells

Zinc transporter ZIP8 (SLC39A8) and zincinfluence IFN-� expression in activated

human T cellsTolunay B. Aydemir,*,† Juan P. Liuzzi,* Steve McClellan,‡ and Robert J. Cousins*,†,1

*Center for Nutritional Sciences, College of Agricultural and Life Sciences, †Department of Biochemistry and MolecularBiology, College of Medicine, and ‡Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville,

Florida, USA

RECEIVED DECEMBER 22, 2008; REVISED MARCH 26, 2009; ACCEPTED MARCH 27, 2009. DOI: 10.1189/JLB.1208759

ABSTRACTThe zinc transporter ZIP8 is highly expressed in T cellsderived from human subjects. T cell ZIP8 expressionwas markedly up-regulated upon in vitro activation. Tcells collected from human subjects who had receivedoral zinc supplementation (15 mg/day) had higher ex-pression of the activation marker IFN-� upon in vitro ac-tivation, indicating a potentiating effect of zinc on T cellactivation. Similarly, in vitro zinc treatment of T cellsalong with activation resulted in increased IFN-� ex-pression with a maximum effect at 3.1 �M. Knockdownof ZIP8 in T cells by siRNA decreased ZIP8 levels innonactivated and activated cells and concomitantly re-duced secretion of IFN-� and perforin, both signaturesof activation. Overexpression of ZIP8 by transienttransfection caused T cells to exhibit enhanced activa-tion. Confocal microscopy established that ZIP8 is lo-calized to the lysosome where ZIP8 abundance is in-creased upon activation. Loss of lysosomal labile zinc inresponse to activation was measured by flow cytom-etry using a zinc fluorophore. Zinc between 0.8 and 3.1�M reduced CN phosphatase activity in a linear man-ner. CN was also inhibited by the CN inhibitor FK506and ZIP8 overexpression. The results suggest that zincat low concentrations, through inhibition of CN, sustainsphosphorylation of the transcription factor CREB, yield-ing greater IFN-� expression in T cells. ZIP8, throughcontrol of zinc transport from the lysosome, may pro-vide a secondary level of IFN-� regulation in T cells. J.Leukoc. Biol. 86: 000–000; 2009.

IntroductionZinc homeostasis in cells, including those of the immune sys-tem, is maintained through tight regulation of zinc influx, ef-flux, and distribution to intracellular organelles. Zinc trans-

porter proteins are essential for these metabolic and func-tional adjustments. Zinc transporters are found within twogene families: the ZIP (SLC39) and ZnT (SLC30) [1, 2]. ZIPtransporters function in zinc influx into the cytosol, and ZnTtransporters function in zinc efflux from the cytosol.

Zinc has a variety of effects on the immune system in vivoand in vitro. These effects are mainly concentration-dependent[3]. For example, zinc increases proinflammatory cytokinemRNA levels in monocytes in vitro and also exhibits a biphasiceffect on cytokine expression as zinc levels are increased [4].IFN-� production by PHA-stimulated T cells was decreased inexperimental human zinc deficiency [5]. Similarly, reducedIFN-� production in response to presentation of the Heligmo-somoides polygyrus antigen was identified in zinc-deficient mice[6]. These findings suggest that immune cells require a finiteamount of zinc to function optimally.

T cells play one of the central roles in adaptive immunity.Upon activation through the TCR, T cells mediate the im-mune response by secreting cytokines or a granule exocytosis-mediated cytotoxic mechanism [7]. The cytokine, IFN-�, is ahallmark of T cells, and it is crucial for immunity against intra-cellular pathogens and for tumor suppression. IFN-� expres-sion is controlled by specific transcription factors in activatedT cells. Among them, CREB regulates IFN-� production posi-tively in human T cells through binding to the IFN-� pro-moter [8], which is enhanced by CREB phosphorylation [9]. Ithas been shown that CREB is dephophorylated by CN in manycell types including pancreatic islet cells and neurons [10],suggesting that CREB might be dephosphorylated by CN in Tcells as well. CN phosphatase activity is inhibited by zinc invitro in a concentration-dependent manner [11, 12]. There-fore, zinc may have a role in increased IFN-� expressionthrough inhibition of CN during T cell activation.

The granule exocytosis-mediated cytotoxicty pathway is usedby CD4 and CD8 T cells [7]. Upon TCR activation, T cellsgain the ability to execute granule exocytosis. Degranulationreleases the pore-forming protein perforin and several pro-

1. Correspondence: University of Florida, 201 FSHN Building, P.O. Box110370, Gainesville, FL 32611, USA. E-mail: [email protected]

Abbreviations: AC�adenylate cyclase, CN�calcineurin, hZIP8�humanZIP8, LAMP1�lysosome-associated membrane protein, LAT�latency-as-sociated transcript, MFI�mean fluorescence intensity, MT�metallothionein,MTF-1�metal-regulatory transcription factor 1, PKA�protein kinase A,qPCR�quantitative PCR, siRNA�small interfering RNA

Article

0741-5400/09/0086-0001 © Society for Leukocyte Biology Volume 86, August 2009 Journal of Leukocyte Biology 1

Uncorrected Version. Published on April 27, 2009 as DOI:10.1189/jlb.1208759

Copyright 2009 by The Society for Leukocyte Biology.

teases or granzymes. Perforin is secreted from lysosomes toform pores, allowing proteases and granzymes to enter to thetarget cells and induce apoptosis. Pore formation is a calcium-dependent process, and it is inhibited by zinc [13], suggestingthat the concentration of lysosomal zinc is an important com-ponent for effective perforin function during T cell activation.

Relative to the involvement of zinc in immune cells, thetransporter protein ZIP8 has been shown by Begum et al. [14]to be highly expressed in human immune-stimulated mono-cytes and differentiated macrophages. In transfected cells, itwas localized to the lysosome and was inducible by immunestimuli such as LPS. Those findings support the 5.6- to 9.8-foldup-regulation of ZIP8, which we identified within a dataset de-rived from a microarray analysis of RNA from PBMCs of hu-mans that were infused with LPS [15]. Furthermore, we haveshown previously that ZIP8 is highly expressed in purified hu-man T cells compared with monocytes or granulocytes [16].Based on the high ZIP8 expression in human T cells and thatzinc supplementation of human subjects produces enhancedIFN-� expression upon activation in vitro [16], we have exam-ined the relationship of ZIP8 expression further to a processthat appears to potentiate activation of human T cells.

Here, we used CD3� circulating primary human T cells toconduct our experiments. Our data demonstrate that ZIP8 isup-regulated during T cell activation. ZIP8 protein was local-ized in the lysosome, and ZIP8 transports zinc from the lyso-some to cytoplasm of T cells during TCR-mediated activation.The increased cytoplasmic zinc caused inhibition of CN activ-ity, resulting in mild increases in phosphorylated CREB andproduction of IFN-�, and the decreased lysosomal zinc causedan increase in perforin secretion. These experiments relateZIP8 expression/function directly to the TCR-mediated T cellactivation process.

MATERIALS AND METHODS

Human subjectsHealthy male subjects (21–31 years old) were used for peripheral bloodcollection after 4 days of zinc supplementation for the assessment of theeffect of zinc on T cell activation. During this study, each subject con-sumed a zinc supplement (15 mg Zn as ZnSO4) or a placebo. For themechanistic experiments, other healthy male subjects, who were not zinc-supplemented, provided samples of peripheral blood. Procedures used in-formed consent and had the approval of the University of Florida Institu-tional Review Board (Gainesville, FL, USA).

Isolation and TCR-mediated activation of T cellsWhole blood was mixed with PBS and was layered on Histopaque 1.077(Sigma Chemical Co., St. Louis, MO, USA) and centrifuged at 600 g atroom temperature for 30 min. The PBMCs at the interface were removedand washed two times with PBS using 250 g for 10 min. The cells were sus-pended in MACS buffer (PBS, pH 7.2, with 0.5% BSA and 2 �M EDTA)and mixed with magnetically labeled microbeads (Miltenyi Biotec, Ger-many) conjugated to a cocktail of antibodies against nonlymphocytic cellsfor isolation of T cells through negative selection. Flow cytometry-demon-strated purity of CD3� T cells was �95%. Isolated T cells were plated at adensity of 2 � 106 cells/ml in X-VIVO15� medium (Cambrex, Charles City,IA, USA) containing 5% human AB serum in 48-well plates. This formula-tion produced a medium with a zinc concentration of 1.2 �M. In some ex-periments, zinc was added (as zinc sulfate) to increase the zinc concentra-

tion of the medium incrementally by between 1.6 and 25 �M. For TCR ac-tivation, the T cells were incubated for 48 h at 37°C in 5% CO2 withmicrobeads conjugated to antibodies against human CD2, CD3, and CD28(Miltenyi Biotec). These methods, which mimic antigen presentation, havebeen described previously [16]. For the assessment of the effect of zinc invitro, cells were pretreated with indicated concentrations of zinc sulfate for2 h.

Overexpression and siRNA-mediated suppression ofhZIP8 expressionThe T cells were plated at a density of 2 � 106 cells/ml with medium(Cambrex) as above. For hZIP8 overexpression, the cells were transientlytransfected with pCMV-Sport6 containing full-length hZIP8 cDNA (Ori-Gene, Rockville, MD, USA) or empty pCMV-Sport6 using Effectene reagent(Qiagen, Valencia, CA, USA). Activation was initiated 2 h after transfection.Cells were harvested 48 h after activation was initiated. For ZIP8 knock-down, the cells were plated at a density of 1 � 106 cells/ml and were trans-fected using Hyperfect reagent (Qiagen) with 750 ng hZIP8 SMARTpoolsiRNA (Dharmacon, Boulder, CO, USA) or nontargeting random siRNA(Dharmacon). After 16 h, fresh medium was added, and cells were acti-vated as above. Cells were harvested after a 48-h activation period. Cell via-bility, as measured by trypan blue exclusion, was not influenced by thesetreatments.

RNA isolation and qPCR analysisTotal RNA was isolated from the purified T cells using TRIzol reagent (In-vitrogen, Carlsbad, CA, USA). To protect against residual DNA contamina-tion, all RNA samples were treated with Turbo DNA-free reagents (Am-bion, Austin, TX, USA) as described by the manufacturer. Primers andTaqMan probe sequences used for qPCR have been reported [16, 17]. Rel-ative quantitation for all assays used 18S rRNA as the normalizer.

Antibody productionA polyclonal rabbit antibody against hZIP8 was raised to a peptide (FGND-NFGPQEKT) selected from the full-length sequence [14]. A cysteine resi-due was added to the N terminus for coupling to the carrier protein andfor conjugation to Sulfolink (Pierce, Rockford, IL, USA) for affinity purifi-cation. This antibody was prepared in rabbits using methods we have de-scribed previously [18].

Confocal laser-scanning microscopy and flowcytometryAfter 48 h in culture, the cells were fixed in 2% paraformaldehyde in PBSand incubated with 0.1% Triton X-100 for permeabilization. Immunolabel-ing was with the anti-hZIP8 polyclonal antibody or goat anti-LAMP1 anti-body (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Secondary label-ing was performed with Alexa Flour 594, conjugated to anti-rabbit IgG andAlexa Flour 488 (Invitrogen), conjugated to anti-goat IgG, respectively.Nonactivated cells were also incubated with LysoTracker (Invitrogen) andthe zinc-specific fluorophore, FluoZin-3 (Invitrogen), to examine labile zinclocalization. Imaging was with a Leica TCS SP5 laser-scanning confocal mi-croscope with LAS-AF imaging software, using a 40� oil objective. For de-tection of labile zinc by flow cytometry, cells were incubated with FluoZin-3for 30 min. After an additional 30-min incubation in serum-free mediawithout fluorophore, cells were washed, and fluorescence was measured.This was followed by addition of 100 �M ZnCl2 for 90 s, and fluoresencewas measured again.

SDS-PAGE and Western analysisMembrane proteins were isolated using a specialized protein extractionmethod (BioVision, Mountain View, CA, USA) prior to PAGE. To confirmequivalent loading, protein bands were visualized by Ponceau staining aftertransfer to nitrocellulose membranes. Alternatively, total cell lysates were

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analyzed. For these preparations, cells were placed in a buffer containing1% Triton X-100, 50 �M Tris HCl (pH 7.4), and protease inhibitor (SigmaChemical Co.) and sonicated for 10 s. Actin (Sigma Chemical Co.) wasused as the loading control. Western blots were produced as described pre-viously [18] with the affinity-purified rabbit anti-hZIP8 antibody above. Im-munoreactivity was visualized by chemiluminescence (Pierce) and X- rayfilm. Band intensities for upper and lower bands of ZIP8 and loading con-trols were quantified by using GenTools (SynGene, UK) software.

Perforin and cytokine assays and T cell TCR signalinganalysisT cells were removed by centrifugation, and the perforin concentration ofthe medium was measured using a sandwich ELISA (Diaclone). IFN-� se-cretion into the medium was measured with Beadlyte human 26-plex multi-cytokine detection reagents (Millipore, Bedford, MA, USA). Fluorescencemeasurements using a Luminex 200 system were normalized for total inputprotein in the medium. Cells were lysed with Beadlyte universal buffer(Millipore). Similarly, the relative abundance of phospho-CD3 (phosphoty-rosine), phospho-Lck, phospho-ZAP70, phospho-LAT, and phospho-CREBin cell lysates was assessed with the Beadlyte phosphoprotein detection sys-tem (Millipore). Fluorescence measurements using a Luminex 200 systemwere normalized for total input protein in total cell lysate and expressed asrelative MFI.

Phosphatase activity of CN was measured at the cellular level. The celllysates were obtained by ultracentrifugation (150,000 g) and desalted withresin columns. In the CN activity assay, calmodulin and the cell lysate wereincubated with zinc or the CN inhibitor FK506 (A.G. Scientific, San Diego,CA, USA) for 10 min at 30°C. The reaction was initiated by addition ofsubstrate and terminated by addition of malachite green. Phosphate releasefrom the substrate was measured spectrometrically (Calbiochem, San Di-ego, CA, USA).

Statistical analysisData are expressed as means � sd and were analyzed by one-way or two-way ANOVA (as appropriate) followed by the Student-Newman-Keuls multi-ple comparisons test or as otherwise indicated in figure legends. Student’st-test (two-tailed) was used for analysis between two groups. Minimal statisti-cal significance was set at P � 0.05. Data are representative of at least threeindependent experiments or as otherwise indicated in figure legends.

RESULTS

Zinc influences T cell activation in human subjectsActivation of primary T cells from the human subjects pro-duced increases in IFN-� transcripts (Table 1). Activation re-sponses were enhanced significantly when an oral zinc supple-ment was provided to the subjects from whom the cells were

isolated. Using qPCR, relative transcript levels for zinc trans-porters in RNA from these cells were evaluated. ZIP8 mRNAwas the most up-regulated upon activation (Fig. 1). Althoughnumerous zinc transporters are expressed in these cells, in ad-dition to ZIP8, only ZIP3 and ZIP14 transcripts were elevatedsignificantly upon activation. Overall, the data show that inhuman subjects, T cell ZIP8 is highly up-regulated during im-mune activation, and activation is potentiated by zinc con-sumption. These data provided a focus for the following stud-ies.

T cell activation increases ZIP8 expressionAn increase in expression of a zinc transporter in an immunecell upon activation suggests zinc dependency for some activa-tion-related process. As shown in Figure 2, activated T cells aresensitive to zinc added in vitro. Expression of IFN-� mRNA wasbiphasic with a maximum at 3.1 �M Zn (Fig. 2A). For compar-ative purposes, we also examined the zinc responsiveness ofMT, a gene that is not a specific marker for activation but isan indicator of cellular zinc status. Particularly relevant is thatMT mRNA increased as a progressive trend, significant onlywhen the zinc concentration of the medium was 6.2 �M orabove (Fig. 2B). That extracellular zinc concentration suggestsit yields an intracellular zinc concentration-threshold necessaryfor activation of the MTF-1. Also relevant is the extreme sensi-tivity of IL-2 mRNA levels to extracellular zinc (Fig. 2B). Thisobservation suggests that a pathway leading to IL-2 expressionis inhibited strongly by zinc. That IFN-� mRNA is maximal at3.1 �M suggests that expression of the gene is sensitive to lowbut restricted concentrations of extracellular zinc and lessthan that required for MTF-1 activation.

To investigate changes in ZIP8 protein in response to activa-tion, we used Western blot analysis with the polyclonal hZIP8

TABLE 1. Influence of Supplemental Zinc on Activation of TCells from Human Subjects

Activation Zinc IFN-� mRNA

– – 1 � 0.05a

– � 1.3 � 0.4a

� – 107 � 16b

� � 197 � 63c

Cells were obtained from subjects who were given zinc (15 mg/day)or a placebo for 4 days. The cells were activated for 2 days in vitro.Data are expressed as means � sd, n � 3, and were analyzed by two-way ANOVA. Values with a different superscript are statistically differ-ent (P�0.05�P�0.001).

Figure 1. Activation of T cells changes zinc transporter expression.Relative ZnT and ZIP transcript levels in total RNA from activated andnonactivated primary human T cells were measured by qPCR. Data areexpressed as relative to the nonactivated control and are normalizedto 18S rRNA. Representative data are presented. Values shown aremeans � sd (n�4). *, P � 0.01; **, P � 0.001, compared with nonac-tivated control.

Aydemir et al. Zinc transporter ZIP8 and T cell activation

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antibody described above. There were two bands, �75 and�150 KDa, detected by SDS-PAGE upon activation. To testspecificity, the ZIP8 antibody was preincubated with the pep-tide antigen, and as shown in Figure 3A, both bands wereeliminated. PNGase treatment was used to test if ZIP8 proteinwas glycosylated. We did not observe any reduction in eitherband with this treatment (data not shown). These two bandswere also detected by native gel electrophoresis (data notshown). These data may suggest collectively that in activated Tcells, ZIP8 may exist in two forms: a monomer and a dimer.The increase in intensity of both bands in total membrane andtotal cell lysate preparations is shown in Figure 3B. Ponceaustaining was used as the loading control for membrane prepa-rations. Actin was used as a loading control for total cell lysate,and the bar graphs represent the quantification of both ZIP8band intensities of the two bands after normalization to actin.Densitometry revealed there was a significant increase in ZIP8

protein produced upon T cell activation. In agreement withthe Western blot analysis, activation increased ZIP8 abundance(red) markedly, as visualized by confocal microscopy (Fig. 3C).Preincubation of the antibody with the ZIP8 peptide antigencaused the disappearance of ZIP8 fluorescence intensity (Fig.3C). These data show collectively that activation of T cells in-creases ZIP8 synthesis.

It has been reported previously that hZIP8 localizes to lyso-somes in ZIP8-transfected human embryo kidney cells [14]. Toinvestigate ZIP8 localization in activated human primary Tcells, we used the same polyclonal ZIP8 antibody (red) asabove and an antibody against a lysosome-specific membranemarker, LAMP1 (green). Confocal images showed a colocaliza-tion between ZIP8 and LAMP1 (Fig. 3D). The localization ofLAMP1 to the lysosome shown here is similar to that reportedby Shen et al. [7]. This localization target (lysosome) is sup-ported by the images presented in Figure 4C using the lysoso-mal probe, LysoTracker. Of particular note is the prevalenceof yellow when the individual images are merged (Fig. 3D).Colocalization of ZIP8 and LAMP1 upon activation suggeststhat ZIP8 might function to transport zinc from lysosomal vesi-cles to the cytoplasm.

To investigate labile zinc transitions during activation, weused the highly zinc-specific fluorophore FluoZin-3. The cellswere placed in serum-free medium containing FluoZin-3 for 30min and then in the medium without the fluorophore for anadditional 30 min. Flow cytometry was used to monitor fluores-cence intensity produced when FluoZin-3 interacts with labilezinc (Fig. 4A). Note that in the upper two panels, only back-ground fluorescence (0–4%) is produced with control andactivated cells. The lower two panels show fluorescence 90 safter 100 �M zinc is added in vitro to the same cells. This invitro addition of zinc is necessary to provide an influx of zincions required to bind to FluoZin-3 and produce fluorescence.Of note is the significant fluorescence produced by the con-trol cells. This is shown through the amount of fluorescence(93%) appearing in the FITC channel. Virtually no fluores-cence is found with activated cells. We propose that this differ-ential occurs, as control cells have low levels of ZIP8 and donot transport labile zinc from lysosomes. In contrast, activatedcells have high ZIP8 abundance and export zinc rapidly to thecytoplasm. In Figures 2B and 4B, we have shown that there isa robust production of MT in T cells upon activation. Compe-tition studies have shown that MT has an apparent stabilityconstant (4�1011 M–1) for binding zinc at pH 7.4, which is10,000-fold higher than FluoZin-3 (7.1�107 M–1) [19]. There-fore, most likely, the transported zinc produces only a tran-sient increase in cytoplasmic zinc concentrations and is boundto MT rapidly, which quenches the ability of zinc ions to inter-act with FluoZin-3. Nevertheless, it is important to point outthat MT has binding sites for seven atoms of zinc/molecule.Here, we have presented the average apparent stability con-stant of MT for all zinc-binding sites. However, it should betaken into consideration that ions bind to MT (particularly to� cluster) in a manner that allows them to be physiologically(kinetically) active and potentially able to alter a biochemicalprocess [20].

Figure 2. In vitro zinc addition influences the effect of activation onIFN-�, MT, and IL-2 expression in primary T cells. Relative mRNAlevels of IFN-� (A) and MT and IL-2 (B) were measured by qPCR innonactivated and activated cells. The cells were activated after an ini-tial 2-h incubation with the indicated concentration of zinc. Data arenormalized to 18S rRNA and are expressed as relative to the nonacti-vated control that was adjusted to one. Representative data from threeindependent experiments are presented. Values shown are means �sd (n�3–4). P � 0.05–0.001 compared with activated control withoutadded zinc and is indicated by a–d.


Furthermore, we used FluoZin-3 and LysoTracker, a lysoso-mal probe for confocal microscopy, to investigate specific lo-calization of labile zinc in T cells (Fig. 4C). Nonactivated Tcells were used in these experiments for detectable zinc-re-lated fluorescence, as activation caused disappearance of theFluoZin-3 fluoresence signal (as above). Merged confocal im-ages showed an 84% colocalization between FluoZin-3 and Ly-soTracker. The quantitative colocalization was calculated byimaging software. These data obtained by flow cytometry andconfocal microscopies suggest collectively that up-regulatedZIP8 during activation is responsible for the reduction of lyso-somal labile zinc.

ZIP8 knockdown decreases T cell activationTo test whether translocation of lysosomal zinc through theZIP8 transporter would affect T cell activation, we knock down

ZIP8 with siRNA. We verified knockdown of ZIP8 by qPCR andWestern analysis. As shown in Figure 5A, ZIP8 siRNA signifi-cantly reduced ZIP8 mRNA levels �60% in nonactivated cells.Upon activation, the 25-fold increase in ZIP8 mRNA was de-creased 70% with ZIP8 siRNA. Western analysis showed a 55%reduction in ZIP8 upon siRNA transfection (Fig. 5B). BothZIP8 protein bands showed a comparable reduction. The ZIP8siRNA did not produce knockdown of ZIP3 or ZIP14 mRNA(data not shown).

We used enhanced expression of IFN-� mRNA upon Tcell activation to examine the effect of ZIP8 knockdown onactivation. Knockdown caused a 66% decrease in IFN-�mRNA in activated cells, and the basal expression level wasnot affected in nonactivated cells (Fig. 5C). Similarly, secre-tion of IFN-� protein into the culture medium was also di-minished significantly (74%) in activated cells by knock-

Figure 3. ZIP8 protein is up-regulated in response to primary T cell activation and localizes with lysosomes. Western blot analysis (A and B) andconfocal images (C and D) from primary T cells. Total membrane proteins and total cell lysates were isolated and used for SDS-PAGE. Blots wereprobed with an affinity-purified polyclonal hZIP8 antibody. (A) Blots were probed with affinity-purified, polyclonal hZIP8, directly or after preincu-bation with its peptide antigen (Pept). Act, Activated cell. (B) Representative Western blots from three independent experiments for membraneproteins and total cell lysates. The average values of both ZIP8 band intensities from three independent experiments are shown. Values shown aremeans � sd (n�6); *, P � 0.02: **, P � 0.001, compared with nonactivated control (Cont). (C) Immunofluororescence localization of ZIP8 (red)by laser-scanning confocal microscopy. Nonactivated and activated primary T cells probed with the ZIP8 antibody. The area in white boxes repre-sents magnified images. (D) Immunofluororescence localization of ZIP8 (red) and LAMP1 (green), a lysosome-specific membrane marker, in acti-vated T cells by laser-scanning confocal microscopy. The area that was chosen for enlarged images was indicated by white squares. Yellow indicatesthe extent of colocalization.



down of ZIP8 (Fig. 5D). To further establish the relation-ship of T cell activation, lysosomal zinc export, and ZIP8,we measured secretion of perforin. The specific interest inthis protein was because its secretion from lysosomes is acharacteristic of activated T cells. Importantly, it has beenshown that zinc inhibits pore formation [13], leading us tohypothesize that reduction in lysosomal labile zinc might benecessary for optimum lysosomal secretion in T cells duringactivation. As is shown in Figure 5E, siRNA knockdown ofZIP8 caused a 30% reduction in perforin secretion by acti-vated cells. By contrast, in companion experiments, overex-pression of ZIP8 caused a 32% increase in perforin secre-tion in activated T cells (Fig. 5F). In Figure 5, D–F, theIFN-� and perforin data are presented on a per mg proteinin medium basis to normalize for differences in cell prolif-eration and/or changes in secretory patterns. Collectively,these data show that ZIP8 is necessary for potentiatingIFN-� production and perforin secretion. These results pro-vide further evidence that a reduction of labile zinc in lyso-somes, as shown by FluoZin-3 fluorescence, through up-reg-ulation or overexpression of ZIP8 in T cells, enhances thesecretion of perforin, and inhibition of ZIP8 causes a reduc-tion in secretion.

ZIP8 and zinc stimulate T cell activationZIP8 is up-regulated during T cell activation (Figs. 1 and2B). To simulate high expression of ZIP8, we transientlytransfected T cells with the ZIP8 plasmid as above (Fig. 5F).

There was a 20-fold increase in ZIP8 mRNA and 67% in-crease in ZIP8 protein levels 48 h after transfection (Fig. 6,A and B). Cells that were transfected and then activatedhad the highest expression of ZIP8. To test the effect ofZIP8 overexpression on activation, we measured IFN-�mRNA and protein level (Fig. 6, C and D). The increase ofIFN-� mRNA and protein produced by activation was ampli-fied further when the cells were transfected with ZIP8cDNA. This shows a direct influence of ZIP8 on activation.In the same experimental setting, we also included low andhigh zinc conditions to investigate the possibility that ZIP8is influencing IFN-� production through a transient alter-ation of cytoplasmic zinc concentrations. Addition of 3.1�M zinc enhanced IFN-� expression to the same extent asZIP8 overexpression, and 25 �M zinc diminished the en-hanced expression of IFN-� (Fig. 6, C and D). We have alsoanalyzed expression of human MT mRNA by qPCR in ZIP8-overexpressing cells. We found a 3.6-fold increase (data notshown). That finding confirms that overexpression producesincreased cytosolic zinc in an amount sufficient to initiateMT synthesis. Overall, the data from Figure 6 show thattransfection with ZIP8 yields the same level of enhancementin IFN-� expression as is achieved through in vitro additionof 3.1 �M zinc during T cell activation (Fig. 2). These dataemphasize collectively that a small increase in cytoplasmiczinc provided through the translocation of zinc from lyso-some by ZIP8 contributes to enhanced expression of IFN-�.

Figure 4. Decrease in intracellular-free zinc in activated primary T cells. FluoZin-3 fluoresence was used to assess labile zinc changes in nonacti-vated and activated cells. Flow cytometry experiments were conducted in the absence or presence of 100 �M ZnCl2, which was added for 1.5 min(A). Fluorescence intensity (green) was examined by flow cytometry. T cells were activated for 48 h and probed with MT antibody (blue). Imagesfrom nonactivated and activated cells were obtained by laser-scanning confocal microscopy. (B) The area in white boxes represents magnified im-ages. Confocal microscopy was used to visualize localization of FluoZin-3 and lysosomes (C). After initial incubation with FluoZin-3, nonactivated Tcells were probed with LysoTracker (red), and colocalization (yellow) with free zinc was shown in merged images.


Zinc acts on CREB via CNWe investigated which signaling pathway influencing IFN-�production in response to TCR activation could be zinc-depen-dent. CD3, Lck, Zap70, LAT, and CREB proteins were phosho-rylated in response to TCR activation for 48 h (Fig. 7A). Ofnote is that in contrast to Western analysis, which measuresthe extent of phosphorylation in one time-point, the Luminex-based assay used in these experiments provides a cumulativemeasure of phosphorylation, as the beads containing the spe-cific antibodies are present throughout the 48 h of incubation.The data are normalized for total protein in each sample. Ofnote, only CREB phosphorylation was affected by in vitro addi-tion of zinc. Although the preincubation of activated cells with3.1 �M zinc increased CREB phosphorylation significantly, itwas diminished by 25 �M zinc and returned to the basal level.It has been shown in many cell types that the Ca/calmodulin-regulated phosphatase CN dephosphorylates CREB [10]. Zinc

also inhibits CN phosphatase activity [11, 12]. To test for aconcentration-dependent effect of zinc on CN, we used an invitro CN assay that measures phosphatase activity (data notshown). The results showed that CN activity was inhibited byzinc in a concentration-dependent manner. At the zinc con-centration 3.1 �M, CN activity was inhibited (90%). Furtherevidence that zinc influences CN activity specifically in T cellswas obtained by using a cellular CN activity assay (Fig. 7B).The results showed that CN activity was increased upon activa-tion. When the cells were treated with 10 �M FK506 or 3.1�M zinc along with activation, CN activity was inhibited (Fig.7B). We also measured CN activity at 25 �M zinc, and the in-hibition was the same as that observed with 3.1 �M zinc (datanot shown). To test the effect of ZIP8 on CN activity, we over-expressed ZIP8 in T cells along with activation. Compared withthe activated T cells, those overexpressing ZIP8 showed de-creased CN activity (Fig. 7B). These results suggest that ZIP8

Figure 5. ZIP8 knockdown with siRNA decreases IFN-� and perforin production inactivated T cells. After transfection with ZIP8 siRNA or nontargeting random siRNAfor 16 h, the T cells were activated for 48 h. Transcript levels of ZIP8 (A) and IFN-�(C) were measured by qPCR. Representative data from three independent experi-ments are expressed as relative to nonactivated control and normalized to 18SrRNA. Values shown are means � sd (n�3). (B) Representative Western blot oftotal cell lysates from siRNA-transfected, activated T cells. The average values ofboth ZIP8 band intensities from three independent experiments are shown. Valuesshown are means � sd (n�6). (D) Secretion of IFN-� into the medium was mea-sured by Luminex multicytokine detection. Representative data are expressed as rel-ative to control and were normalized to total protein in the medium. (E and F) Se-cretion of perforin into the medium was measured by ELISA and normalized to to-tal protein in the medium. Representative data from three independentexperiments are shown for measurement of secreted perforin with ZIP8 knockdown(E) or overexpression (F) conditions. Values shown are means � sd (n�3) in non-activated cells compared with activated cells and transfected with ZIP8 siRNA orpCMV6-ZIP8. (A, C–F) Bars with a–d are different (P�0.001–0.003).



influences IFN-� expression via inhibition of CN through achange in the cytoplasmic zinc concentration. Finally, we usedthe FK506 inhibitor to test whether CN influences CREB de-phosphorylation in T cells by using the Beadlyte phospho-pro-tein detection system. When CN activity was inhibited byFK506, CREB phosphorylation was increased further (Fig. 7C).

DISCUSSION

ZIP transport proteins are believed to control intracellular zincconcentrations in mammalian cells through transport acrossthe plasma membrane or from intracellular vesicles/organelles[2]. The net result is an increase in zinc in the cytoplasm. It ispresumed that zinc transport and altered zinc homeostasishave functional outcomes. Our interest in ZIP8 specifically hasbeen stimulated by the relative abundance of ZIP8 transcripts,as markedly higher in T cells than in monocytes or granulo-cytes, respectively [16]. Furthermore, zinc supplementation ofadult human subjects with 15 mg Zn/day, about the recom-mended dietary allowance, increased IFN-� mRNA expressionmarkedly in CD3� primary human T cells upon activation.The high ZIP8 expression in T cells and the evidence that zincactually has a measurable response under realistic dietary con-ditions suggested that T cell activation, a major factor in adap-tive immunity, has a zinc-dependent component. These find-ings led to our hypothesis that ZIP8 has an important role inpotentiating T cell activation.

Faber et al. [21] have also shown the effect of zinc on IFN-�production. When they supplement adult human subjects with80 mg Zn/day (over five times the concentration we used),IFN-� production was reduced. The different effects of 80 mgand 15 mg Zn/day supplementation may simulate the biphasiceffect of zinc on activation that we have shown in our in vitroexperiments. The biphasic effect of zinc on IFN-� expression(Fig. 2) could be the result of activation/inhibition of differ-ent signaling pathways that specifically require different con-centrations of cytoplasmic zinc. For example, CREB enhancesIFN-� expression in human cells when it is phosphorylated.Several phosphatases and kinases have been shown to regulatethe phosphorylation status of CREB. One of the phosphatasesthat is responsible for dephosphorylation of CREB is CN [10].We and others found that CN enzymatic activity is sensitive tozinc. We have found by using an in vitro CN assay that CN ac-tivity was inhibited at concentrations of zinc in the range0.8–25 �M (data not shown). As shown in Figure 2A, 25 �Min vitro addition of zinc inhibited the expression of IFN-�, andCN activity was totally inhibited at that concentration of zinc.These observations collectively support that although there aremultiple mechanisms that regulate the phosphorylation statusof CREB, the influence of zinc is primarily on CN. The poten-tial kinases that are responsible for phosphorylation of CREBare mitogen- and stress-activated kinases, calcium/calmodulin-dependent protein kinase type IV, and PKA [22]. It has beenshown that AC is necessary for the PKA activation, and its en-

Figure 6. Overexpression of ZIP8 enhances T cellactivation. T cells were activated for 48 h aftertransfection with pCMV6-ZIP8 or empty vector for2 h. Transcript levels of ZIP8 (A) and IFN-� (C)were measured by qPCR. Representative data fromthree independent experiments are expressed asrelative to control and normalized by 18S rRNA.Values shown are means � sd (n�3); P � 0.01–0.001 compared with ZIP8-transfected, nonacti-vated cells, indicated by a–d. M, Mock transfection.(B) Representative Western blot of total cell lysatesfrom ZIP8-transfected T cells. The average values

of both ZIP8 band intensities from three independent experiments are shown. Values shown are means � sd (n�6); *, P � 0.001, comparedwith control. (D) Secretion of IFN-� into the medium was measured by Luminex multicytokine detection. Representative data are expressedas relative to nontransfected cells and normalized by total protein in the medium. Values shown are means � sd (n�3); P � 0.01–0.001 com-pared with nontransfected, activated cells as indicated by a–c.


zymatic activity is inhibited by zinc in a concentration-depen-dent manner [23]. Interestingly, 10 �M Zn inhibited total ACactivity in neuroblastoma cells, and lower concentrations didnot have any effect. This suggests that higher concentrationsof cytoplasmic zinc may inhibit IFN-� expression as a result ofinhibition of AC activity and that a modest increase in cyto-plasmic zinc may enhance the phosphorylation of CREB as aresult of inhibition of CN phosphatase activity.

The finding that ZIP3 and ZIP14 are also increased signifi-cantly upon activation may relate to a role in specific T cellfunctions. ZIP3 has been reported as localized to the plasmamembrane of mammary epithelial cells [24]. Knockout of mu-rine ZIP3 causes decreased pre-T cell abundance but onlyupon restriction in dietary zinc [25]. Both observations concurwith a role for ZIP3 in T cell zinc transport. Similarly, we ob-served that ZIP14 expression and localization to the plasmamembrane of murine hepatocytes are IL-6-regulated [26].That observation suggests that there is a cytokine componentto ZIP14 regulation that may contribute to zinc transport dif-ferences across the plasma membrane of T cells. We did notfind any significant change in ZnT transporters in response toT cell activation. ZnT1 was the only known zinc exporter trans-porter that localizes to the plasma membrane. However, it hasbeen shown recently by Overbeck et al. [27] that ZnT4 alsolocalizes on the plasma membrane of T cell line Molt-4. Thisnew finding, however, should be evaluated carefully, as theexpression pattern of proteins might differ between cell linesand primary cells. In our experience, for example, there wasno difference in ZIP8 expression in the Jurkat cell line when

we used the same stimulants as used to activate primary Tcells. Therefore, a specific role of this transporter in responseto TCR-mediated T cell activation in primary T cells needs tobe investigated further.

The predicted molecular weight for ZIP8 is 49.6 KDa. Differ-ent sizes and localizations for ZIP8 protein have been reportedby different groups, however. Initial experiments on ZIP8 werewith monocyte/macrophages. Using uncharacterized antibody,the lysosome was identified as the cellular site for ZIP8, andthe size of the protein was 52 KDa [14]. No functional signifi-cance for ZIP8 was reported; however, Chinese hamster ovarycells transfected with ZIP8 exhibited increased fluorescencefrom Newport Green, suggesting a role in intracellular zincaccumulation. It has been shown by Besecker et al. [28] thatin the human primary lung cells, plasma membrane and mito-chondria were the cellular sites for ZIP8. They detected twobands for ZIP8 protein: �140 and �52 KDa. They also haveshown by FluoZin-3 that ZIP8 was responsible for intracellularzinc accumulation in response to TNF-� treatment. Our datausing FluoZin-3 show that in primary human T cells, the secre-tory lysosome is the site for ZIP8-mediated zinc release. Thesizes of the ZIP8 protein in these T cells as detected by West-ern blot analysis were �150 and �75 KDa. We speculate thatthe 75-KDa protein is newly synthesized, and the 150-KDaband represents the dimer. It has been shown by Lu and Fu[29] that the Escherichia coli YiiP zinc transporter, which be-longs to the cation diffusion facilitator transporter family thatis homologous to the eukaryotic ZnT zinc transporter family,forms a dimer in the presence of zinc ions. Therefore, we ex-

Figure 7. Zinc enhances CREB phosphorylation via inhibiting CN activity. Phosphorylation of indicated proteinsin the TCR signaling pathway was measured by Luminex phosphoprotein detection (A and C). T cells were acti-vated for 48 h after an initial 2-h incubation with the indicated concentrations of zinc (A) or CN inhibitorFK506 (C). Representative data from three independent experiments are expressed as relative to control andnormalized by total input protein in the cell lysate. Values shown are means � sd (n�3); P � 0.001 comparedwith nonactivated cells, indicated by a–c. p, Phospho. (B) Representative data from three independent experi-ments for cellular CN activity assays are shown. Activity of CN was determined spectrophotometrically based onphosphate release from the substrate. Cell cytosol was obtained from lysates by ultracentrifugation and used fordetection of cellular CN activity. Lysate was incubated with the indicated concentration of zinc or CN inhibitorFK506 for 10 min prior to addition of substrate. Values shown are means � sd (n�3); P � 0.01–0.001 comparedwith control, as indicated by a–c.



tend our speculation that ZIP8 might be functional in a dimer-ized form as is the YiiP zinc transporter. In companion experi-ments, an antibody was produced to mouse ZIP8 by the sameapproach as that used for the antibody to hZIP8 used in thepresent experiments [30]. Of note is that mouse ZIP8 fromRBC membranes had an apparent size of 60 KDa, and ZIP8from erythropoietin-induced differentiating erythroid cells hadtwo bands: 120 KDa (major) and 60 KDa (minor). This agreeswith our interpretation. The specificity of our hZIP8 antibodyis based on the peptide competition shown (Fig. 3A) and com-plete elimination of both ZIP8 bands in ZIP8 siRNA-treatedcells (Fig. 5B). In contrast, in Maden-Darby canine kidneycells, transfected with mouse ZIP8, the transporter was local-ized to the plasma membrane [31]. These differences in local-ization may reflect the markedly different function in the cellsystems under investigation or atypical localization related totransfection.

Numerous signaling pathways have been reported as beingzinc-responsive. It has been proposed that involvement of zincin intracellular signaling can be classified into two categories:The first is the early zinc signaling pathway, where zinc is pro-vided by intracellular sources, and the second is late zinc sig-naling, which is dependent on a transcriptional change in zinctransporter expression [32]. The example of the early zinc-signaling pathway could be CD8 or CD4 interaction with Lckin TCR-mediated T cell activation [33, 34]. Zinc provides theinteraction with cysteine-containing motifs for assembly of theTCR to the T cell signaling protein Lck. This interaction is thefirst step in TCR-mediated T cell activation. We have foundthat there is no change in zinc transporter expression early inthe activation process (first 24 h), suggesting that zinc is pro-vided from intracellular sources (data not shown). It was pro-posed that MT, interacting with oxidizing intracellular condi-tions, is the source of the zinc ions needed for the Lck/TCRassembly process [34]. Previously, we have discussed the link-age of zinc to cellular signaling and interaction with specificintracellular and physiologically regulated ligands such as MT[35]. Expression of Lck has been shown to up-regulate in zinc-depleted mice [36, 37]. This could represent an attempt tocompensate for the lack of zinc binding through overproduc-tion of one of the binding ligands, i.e., Lck.

Downstream of the Lck/TCR interaction, there are manysignaling pathways that are activated in T cells (reviewed inref. [38]). One of the important signaling events is activationof the phospholipase C� and subsequent hydrolysis of phos-phatidylinositol 4,5-bisphosphate. to generate inositol triphos-phate, which causes mobilization of calcium. Increased intra-cellular Ca2� will cause activation of CN. A well-known targetfor CN phosphatase is the NFAT transcription factor. Dephos-phorylated NFAT is necessary for IL-2 expression. However,our results (Fig. 7) suggest that CN is also responsible forCREB dephosphorylation. CREB is necessary for IFN-� expres-sion but in the phosphorylated form. Our specific interest inCN is because of the well-known inhibitory effect of zinc on itsphosphatase activity [39]. As we have shown in Figure 2B, IL-2expression is abolished with even 1.6 �M zinc added to the Tcell cultures, and IFN-� expression is enhanced (Fig. 2A), sug-gesting that at 48 h of activation, zinc has inhibited CN activ-

ity. In the following experiments (Fig. 3), we confirmed thatZIP8 is responsible for an intracellular zinc increase during Tcell activation. Therefore, our results suggest that a zinc-de-pendent process, driven by ZIP8-mediated zinc transport, isresponsible for events that culminate in enhanced CREB phos-phorylation and increased IFN-� expression. As the increase incytoplasmic zinc concentration is caused by a change in a zinctransporter, this effect can be considered as an example of alate zinc signaling pathway.

It has been shown by Haase et al. [40] that intracellular zincincreased immediately after addition of PMA to Jurkat T cells.FluoZin-3 fluorescence was used to monitor immediate mobili-zation of labile zinc in response to PMA. Therefore, it mayserve as another example for the early zinc signaling [32]. Inour work, however, we have chosen to use confocal microscopyand flow cytometry to measure fluorescence, as the focus wasto detect specific localization and function of ZIP8 and labilezinc in T cells at 48 h of activation. It has been shown byMuylle et al. [41] that laser-scanning confocal microscopy pro-vides sufficient spatial resolution to uncover subcellular fluo-rescence patterns. Similar to their data from a fish hepatocytecell line, our confocal microscopy results with FluoZin-3 clearlyshowed that fluorescence produced by labile zinc mostly re-sides in lysosomal compartments in nonactivated T cells. Ourflow cytometry data revealed that there was no detectable fluo-rescence signal in activated T cells at 48 h, as zinc has beentranslocated to cytoplasm by ZIP8 and no longer available forFluoZin-3 interaction. As MT was already high in cytoplasm inresponse to activation, zinc was likely sequestered by MT andother proteins, as at intracellular redox and pH conditions,protein binding is preferred compared with the free zinc tran-sition process, where the metal can react with the FluoZin-3fluorophore.

The mobilization of zinc to a labile pool within immunecells has been the subject of much interest and has been wellreviewed [3, 4, 42]. Many such experiments are performedwith high �25 �M Zn concentrations with frequently variableresults. For intramolecular Zn2� exchanges to occur, oxidizingconditions must be present. The data presented here demon-strate that ZIP8, associated with the lysosomal membrane, mayprovide the conditions of lower than physiologic pH necessaryfor zinc transfer to ligands with regulatory influence. It hasbeen estimated that the labile zinc concentration in unstimu-lated lymphocytes is 0.35 �M [43]. The effect we found with 3�M Zn added in vitro, as a stimulating influence on activation,is within what would be expected with such a basal intracellu-lar zinc concentration. Of particular relevance to the stimulat-ing effect of zinc found in the present report is that 3 �M isthe exact zinc concentration that Huang et al. [11] found re-cently to produce maximal inhibition of CN activity. Also rele-vant to the in vitro effects of zinc is that recently, tyrosinephosphorylation in monocytes is sensitive to low �M concen-trations of zinc, which regulate LPS-induced signal transduc-tion [40]. These effects have yet to be related to a specific zinctransporter.

In conclusion, our results demonstrate that ZIP8 is highlyexpressed in human T cells. ZIP8 expression is up-regulatedmarkedly upon activation. Zinc supplementation of human


subjects enhances subsequent T cell activation in vitro and en-hances ZIP8 expression. Knockdown of ZIP8 expression in pri-mary human T cells using siRNA decreased IFN-� and perforinsecretion, both signatures of activation. As we propose withour model (Fig. 8), ZIP8 localization to the lysosome suggeststhat this transporter potentiates cell activation through intra-cellular zinc translocation. Overexpression of ZIP8 by transienttransfection causes enhanced IFN-� expression and perforinsecretion. The lytic properties of T cells have been related tolysosomal development during activation [7]. The mildly in-creased cytoplasmic zinc produced by ZIP8 inhibits CN phos-phatase activity, resulting in increased CREB phosphorylationand CREB binding to the IFN-� promoter, which simulatesIFN-� synthesis. Although the function of ZIP8 was the focusof the present experiments, the mechanism responsible forup-regulation of ZIP8 upon activation has not been examined.In that regard, it is potentially relevant that hZIP8 has a CREBresponse element within the first 2000 bp upstream from thestart site.

Our findings may also point to the wider involvement ofzinc in signaling that influences T cell development and cell-specific immune functions, particularly those in children andthe elderly [44–48]. The mechanism proposed in this reportmay be related to the success that global zinc supplementationinitiatives have had in reducing morbidity and mortality as aresult of infectious disease in under-nourished populations [3,45, 47].

ACKNOWLEDGMENTS

This research was supported by National Institutes of HealthGrant DK 31127 and Boston Family Endowment Funds. Wealso acknowledge support of the Interdisciplinary Center for

Biotechnology Research and funds from the Bankhead-ColeyCancer Research Program. We thank Mitchell D. Knutson,Fikret Aydemir, Shou-Mei Chang, and Lyle L. Moldawer forhelpful contributions.

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KEY WORDS:signal transduction � adaptive immunity


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Zinc transporter ZIP8 (SLC39A8) and zinc influence IFN- expression in activated human T cells