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The Journal of Immunology, 2005, 175: 4697-4705.
Copyright © 2005 by The American Association of Immunologists

Zinc-Mediated Inhibition of Cyclic Nucleotide Phosphodiesterase Activity and Expression Suppresses TNF-{alpha} and IL-1{beta} Production in Monocytes by Elevation of Guanosine 3',5'-Cyclic Monophosphate1

Verena von Bülow, Lothar Rink and Hajo Haase2

Institute of Immunology, University Hospital, RWTH Aachen University, Aachen, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The trace element zinc affects several aspects of immune function, such as the release of proinflammatory cytokines from monocytes. We investigated the role of cyclic nucleotide signaling in zinc inhibition of LPS-induced TNF-{alpha} and IL-1{beta} release from primary human monocytes and the monocytic cell line Mono Mac1. Zinc reversibly inhibited enzyme activity of phosphodiesterase-1 (PDE-1), PDE-3, and PDE-4 in cellular lysate. It additionally reduced mRNA expression of PDE-1C, PDE-4A, and PDE-4B in intact cells. Although these PDE can also hydrolyze cAMP, only the cellular level of cGMP was increased after incubation with zinc, whereas cAMP was found to be even slightly reduced due to inhibition of its synthesis. To investigate whether an increase in cGMP alone is sufficient to inhibit cytokine release, the cGMP analogues 8-bromo-cGMP and dibutyryl cGMP as well as the NO donor S-nitrosocysteine were used. All three treatments inhibited TNF-{alpha} and IL-1{beta} release after stimulation with LPS. Inhibition of soluble guanylate cyclase-mediated cGMP synthesis with LY83583 reversed the inhibitory effect of zinc on LPS-induced cytokine release. In conclusion, inhibition of PDE by zinc abrogates the LPS-induced release of TNF-{alpha} and IL-1{beta} by increasing intracellular cGMP levels.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Zinc is a trace element that affects many aspects of immune function, including thymic development and the activity of T cells, B cells, and NK cells (1). In particular, zinc has contradictory effects on monocyte activity and the secretion of proinflammatory cytokines. On the one hand, it was shown that zinc stimulates release of the monokines IL-6, IL-1{beta}, and TNF-{alpha} in human PBMC (2) and induces mRNA expression of TNF-{alpha} (3). On the other hand, zinc negatively regulates TNF-{alpha}, IL-8, and IL-1{beta} gene expression in the HL-60 monocyte/macrophage cell line (4). Furthermore, zinc acts synergistically with LPS if applied simultaneously in substimulatory concentrations (5), but zinc pretreatment protects against LPS-induced hepatotoxicity by attenuation of TNF-{alpha} production in liver tissue during liver injury in mice (6).

Inhibition of TNF-{alpha} production can be mediated by cyclic nucleotide signaling. The intracellular level of cyclic nucleotides is regulated by their synthesis by adenylate (AC)3 and guanylate cyclases and their hydrolysis into the corresponding inactive 5'-nucleotide monophosphates by phophodiesterases (PDE). Numerous studies show that selective PDE-4 inhibitors, such as rolipram and Ro-20-1724, attenuate TNF-{alpha} production in human monocytes and PBMC by elevation of the intracellular cAMP concentration (7, 8, 9, 10, 11). The cGMP analogues, dibutyryl cGMP (DB-cGMP) and 8-bromo-cGMP (8-Br-cGMP) were found to attenuate LPS-induced TNF-{alpha} secretion in primary monocytes and murine bone marrow-derived macrophages (8, 12). Eleven distinct families of PDE (PDE-1 to -11) have been identified, most of which include more than one subtype. To date, PDE-1, which hydrolyzes both cGMP and cAMP, cAMP-selective PDE-3, and cAMP-specific PDE-4 have been observed in human monocytes (13, 14). Although an effect of zinc on cyclic nucleotide metabolism in monocytes has not been investigated, zinc inhibits several isoforms of purified recombinant PDE that are present in these cells, including PDE-4A and PDE-3 (15, 16). In PC12 pheochromocytoma cells, an involvement of zinc in cyclic nucleotide metabolism was demonstrated. Zinc treatment of PC12 led to an inhibition of cGMP hydrolysis and caused an elevation of the intracellular cGMP content (17).

The aim of this study was to investigate whether zinc suppresses cytokine release of monocytes via an effect on cyclic nucleotide metabolism, especially by modulating PDE activity. We show that zinc has a dual effect, regulating cellular PDE activity as a direct inhibitor of the catalytic activity of all isozymes of phosphodiesterases expressed in monocytes and additionally as an inhibitor of PDE gene expression. Moreover, zinc-induced inhibition of PDE elevates intracellular cGMP, but not cAMP, and suppresses the LPS-induced production of TNF-{alpha} and IL-1{beta}.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Media and reagents

RPMI 1640 medium, sodium pyruvate, L-glutamine, nonessential amino acids, penicillin, streptomycin, and PBS were purchased from Cambrex. Ficoll was obtained from Biochrom, and FCS was purchased from PAA Laboratories. Chlorpromazine, 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro-20–1724), and quazinone were purchased from Calbiochem, and 8-Br-cGMP, DB-cGMP, cAMP, and cGMP were purchased from Biolog LSI. Isobutyl-1-methylxanthine (IBMX), forskolin (FSK), LPS Escherichia coli serotype 0111:B4, 6-(phenylamino)-5,8-quinolinedione (LY83583), sodium pyrithione, sodium nitrite, L-cysteine hydrochloride, propidium iodide, and N,N,N',N',-tetrakis (2-pyridyl-methyl)-ethylenediamine (TPEN) were obtained from Sigma-Aldrich. Directly before incubation, a 100-mM stock solution of the NO donor S-nitrosocysteine (SNOC) was prepared from L-cysteine hydrochloride and sodium nitrite as previously described (18). All other reagents were purchased from standard sources.

Cell culture

All cell lines were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen and cultured at 37°C in a humidified 5% CO2 atmosphere in RPMI 1640 medium containing 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. The human histiocytic lymphoma cell line U937 was cultured directly in this medium. Media for the human acute monocytic leukemia cell lines THP-1 and Mono Mac1 were additionally supplemented with 5 µl/L 2-ME (THP-1) or with nonessential amino acids and 1 mM sodium pyruvate (Mono Mac1).

Isolation and culture of PBMC and monocytes

PBMC were isolated from heparinized peripheral venous blood from healthy donors by centrifugation over Ficoll-Hypaque, washed three times with PBS, and resuspended in RPMI 1640 medium (containing 10% heat-inactivated FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine). For enrichment of monocytes, PBMC were seeded in 100-mm diameter plastic culture dishes at a density of 2 x 106 cells/ml and were incubated for 1 h at 37°C in 5% CO2. Nonadherent cells were removed by washing with medium at 37°C. The monocyte preparations contained 80% CD14+ cells, as determined by flow cytometry.

Flow cytometric analysis

Mono Mac1 cells and PBMC were seeded at a density of 1 x 106 cells/ml. After incubation with zinc and pyrithione for 24 h, 1 x 106 cells were suspended in PBS (1 ml). PBMC were stained with 0.5 µg of FITC-conjugated anti CD14 mAbs or isotype-matched control IgG2b (BD Pharmingen) for 20 min at room temperature to label the monocytes. PBMC and Mono Mac1 cells were stained with propidium iodide (10 µg/ml) for 20 min at 4°C in the dark and analyzed by flow cytometry with a FACSCalibur (BD Biosciences).

Quantification of cytokines

Before the incubations specified in the figure legends, cells were seeded at a density of 5 x 105 cells/ml in 24-well tissue culture dishes for 1 h (PBMC/monocytes) or in six-well tissue culture dishes overnight (Mono Mac1). After the incubations, culture supernatants were harvested and stored at –80°C. For measurement of cellular cytokine content, cells were washed twice with ice-cold PBS; suspended in 50 mM Tris-HCl, pH 7.4, containing 1% (w/v) Triton X-100, 300 mM NaCl, 5 mM EDTA, and 0.02% (w/v) sodium azide; incubated on ice for 15 min; lysed by sonification; and stored at –80°C. For quantification of TNF-{alpha} and IL-1{beta}, ELISAs (BD Pharmingen) were used. Results were measured with an ELISA plate reader (Sunrise; Tecan).

RT-PCR

For investigation of PDE subtype expression, Mono Mac1, U937, and THP-1 were cultured routinely in 25-cm2 plastic flasks. RNA of PBMC and monocytes was extracted 1 h after isolation. For the investigation of changes in PDE mRNA expression, Mono Mac1 cells (3 x 105/ml) were cultured in six-well tissue culture dishes overnight before treatment with zinc and/or LPS. For all samples, total RNA was isolated using the RNeasy kit (Qiagen), and cDNA was prepared using the Reverse Transcription System (Promega) according to the manufacturers’ instructions. PCR was performed using HotStarTaq DNA polymerase (Qiagen) in a final volume of 50 µl using 50 ng/µl cDNA with the following cycle parameters: 95°C for 15 min, 35 cycles of denaturation at 95°C for 40 s, annealing at 60°C for 1 min, and extension at 72°C for 3 min, with a 5-min extension at the last cycle. {beta}-Actin was used as a housekeeping gene. In the case of PDE-1B, PDE-3B, and PDE-4B, 31 cycles of amplification were used, and TNF-{alpha} gene products were amplified in 28 cycles. Primers (TIB Molbiol) were added at a final concentration of 1 µM (PDE), 0.8 µM (TNF-{alpha}), 0.15 µM (IL-1-{beta}), and 0.1 µM ({beta}-actin).

Oligonucleotide primers for PDE-1 and -4 subtypes were used as previously reported (19). Primer for PDE-3 subtypes were designed according to sequences derived from GenBank: PDE-3A, 5'-TTCAACCAGTGATTCAGATTCTGACAGTGG-3' and 5'-CAGTACTAAGAGCAAAAGCTGCTTAGTTGG-3' (accession no. M91667); and PDE-3B, 5'-TATCACAATCGTATACATGCCACAGA-3' and 5'-TTTAAGATCCGTAGCAAGGTTGC-3' (accession no. NM 000922.2). Primers for {beta}-actin (20), TNF-{alpha} (21), and IL-1{beta} (22) were used as previously described. Human prostate cDNA (gift from Dr. M. V. Cronauer, Heinrich-Heine University, Dusseldorf, Germany) and human colon cDNA (Stratagene) served as positive controls (19). After PCR amplification, 10 µl of each sample was separated by electrophoresis in a 1% agarose gel containing 4 ng/ml ethidium bromide.

Determination of PDE activity by HPLC

To measure PDE activity, 1 x 106 Mono Mac1 cells were harvested in sample buffer (25 mM HEPES, 5 mM glucose, 120 mM NaCl, 5.4 mM KCl, 1.3 mM CaCl2, 1 mM MgCl2, and 1 mM NaH2PO4, pH 7.35) and lysed by sonification. The assay was initiated by addition of cGMP or cAMP (final concentration, 50 µM), and incubated for 1 h at 37°C. At the end of the reactions, the remaining cyclic nucleotide concentrations were still present in at least 10-fold excess of the Km of the PDE, ensuring maximum reaction velocity throughout the entire experiment. The reaction was stopped by boiling for 5 min, and any precipitate was removed by centrifugation at 7000 x g for 5 min. Cyclic nucleotides and 5'-mononucleotides were separated by HPLC on a Eurosil C18 column (Knauer) using 25 mM triethylammonium formate, pH 6.5, as an eluent, containing 8 or 4.5% methanol for cAMP or cGMP, respectively. Absorption was measured with an A0293 variable wavelength detector (Knauer) at 258 nm (cAMP) or 252 nm (cGMP). Data were acquired and processed with Eurochrom 2000 chromatography software (Knauer), and PDE activity was calculated as the decrease in peak area for the cyclic nucleotide. The concentration of endogenous cyclic nucleotides in the lysate was too low to be detected with this method and can be disregarded.

Measurement of cellular cAMP

For analysis of cellular cAMP, 4 x 106 Mono Mac1 cells were seeded in polypropylene tubes in 1 ml of culture medium. After incubation at 37°C and 5% CO2, the medium was removed by centrifugation for 5 min at 7000 x g. The cellular cAMP level was detected using the Delfia cAMP kit (PerkinElmer), a time-resolved fluoroimmunoassay based on europium dissociative fluorescence enhancement. The assay was performed according to the manufacturers’ instructions with some modifications. Samples were treated with 4 mM IBMX immediately before addition of Delfia lysis buffer and sample buffer and were incubated for 10 min at room temperature. The cAMP assay plate was incubated with anti-cAMP serum for 30 min. Anti-cAMP serum was removed with three washing steps using Delfia wash buffer. Fifty microliters of standards and samples were added to the plate and incubated for 30 min with gentle shaking. The Eu-cAMP tracer solution was added and incubated for an additional 30 min. After four washes, enhancement solution was added and incubated for 5 min at room temperature. The plates were read using a Tecan Ultra 384 fluorometer with the following setup: 340-nm excitation filter; 612-nm emission filter; counting delay, 400 µs; and counting window, 400 µs.

Measurement of cellular cGMP

For determination of cellular cGMP, Mono Mac1 cells (2.5 x 106 in 1 ml of medium) were seeded in polypropylene tubes and incubated for 6 h at 37°C in 5% CO2. Afterward, cells were pelleted by centrifugation at 3000 x g and 4°C for 5 min and subjected to a purification procedure as previously reported (23). Briefly, cells were lysed by addition of 200 µl of EDTA (30 mM) and boiled for 5 min. Proteins were precipitated by addition of 800 µl of ice-cold ethanol, homogenized for 1 min, and incubated for 30 min at 4°C. Proteins and cell debris were removed by centrifugation at 3000 x g and 4°C for 10 min. The supernatant was evaporated under a nitrogen flow at 56°C, and cGMP in the residue was determined with a cGMP enzyme immunoassay (IBL Hamburg) based on the competition of free cGMP and cGMP bound to acetylcholinesterase, according to the manufacturers’ instructions.

Statistical analysis

Statistical significance of experimental results was calculated by Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
PDE expression in monocytic cell lines and primary cells

Monocytes express three isozymes of PDE: cAMP- and cGMP-hydrolyzing PDE-1 and cAMP-degrading isozymes 3 and 4 (13, 14). The expression profiles of the subtypes of these PDE were analyzed by RT-PCR and compared in human PBMC, primary monocytes, and three monocytic cell lines (Table I). While PDE-1A, PDE-3A, and PDE-4C mRNA were not detected in any cell line or in primary cells, mRNA encoding for PDE-1B, PDE-1C, PDE-3B, PDE-4A, PDE-4B, and PDE-4D were found in PBMC, monocytes, and Mono Mac1 cells. The expression profiles of the remaining two cell lines were similar, except that U937 cells lacked both PDE-3B and PDE-4A, and THP-1 cells did not express PDE-1C. In accordance with these results, the mature monocytic cell line Mono Mac1 was chosen as a model for studying the effect of zinc on PDE activity in monocytes.


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  		Table I. Expression profile of cyclic nucleotide PDE subtypes in monocytic cell lines, PBMC, and primary monocytesa

 
Concentration-dependent effect of zinc ions on TNF-{alpha} release

To establish appropriate conditions for investigation of the inhibitory effect of zinc on LPS-induced secretion of proinflammatory monokines, the release of TNF-{alpha} from Mono Mac1 into the supernatant was quantified by ELISA (Fig. 1A). Cells were stimulated with LPS (250 ng/ml) in the presence of zinc concentrations ranging from 1 to 25 µM together with pyrithione (50 µM), an ionophore that facilitates zinc uptake. Zinc has differential effects on TNF-{alpha} release depending on its concentration (Fig. 1A). Although 1 µM zinc in the presence of pyrithione increased TNF-{alpha} production significantly, 25 µM suppressed the secretion of this cytokine. To elucidate whether the inhibitory effect of zinc depends on the presence of pyrithione, primary monocytes were treated with LPS and zinc without the ionophore. Although higher concentrations were required, zinc inhibited TNF-{alpha} release in the absence of pyrithione (Fig. 1B). To ensure that the reduction in TNF-{alpha} synthesis was not mediated by cytotoxic effects of zinc, membrane integrity was measured with propidium iodide staining, which showed that none of the zinc concentrations used in this paper was toxic (Fig. 1C).



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  		FIGURE 1. Concentration-dependent effect of zinc on LPS-induced TNF-{alpha} release. A, Mono Mac1 cells were stimulated for 4 h with LPS (250 ng/ml) in the presence of zinc concentrations from 1 to 25 µM ZnSO4 plus pyrithione (50 µM). TNF-{alpha} in the cell culture supernatant was determined by ELISA. The values represent the mean ± SEM from three independent experiments performed in duplicate. Statistically significant (p < 0.05) changes in TNF-{alpha} values compared with LPS alone are indicated (*). B, Primary monocytes were incubated with 25 or 125 µM ZnSO4 for 1 h before addition of LPS (250 ng/ml) and incubation for another 24 h. TNF-{alpha} in the cell culture supernatant was determined by ELISA. Values below the lowest standard of 7.8 pg/ml were designated not detectable (n.d.). The values represent the mean ± SEM from four different donors, measured in duplicate. Statistically significant (p < 0.05) differences in TNF-{alpha} values compared with LPS alone are indicated (*). C, Cells were treated as indicated with zinc and/or pyrithione for 24 h. The toxicities of different zinc concentrations were evaluated by FACS analysis after CD14 staining of PBMC and propidium iodide staining (PBMC/Mono Mac1). The values represent the mean ± SEM from four independent experiments (Mono Mac 1) or four different donors (PBMC). Statistically significant (p < 0.05) changes in the survival rate compared with the control values are indicated (*).

 
Inhibition of PDE by zinc ions in cellular lysate

Zinc affects cGMP signaling via inhibition of PDE (17). We investigated whether this effect is specific for cGMP-degrading PDE isozymes or whether zinc acts as a general PDE inhibitor, which also affects the hydrolysis of cAMP. Using selective inhibitors for PDE-1 (chlorpromazine), PDE-3 (quazinone), and PDE-4 (Ro-20-2417), the activity of each isozyme was measured separately in the presence of inhibitors for the other two. To this end, 50 µM cyclic nucleotides were added to Mono Mac1 lysate, and degradation into 5'-monophosphate was monitored by HPLC. The isozymes contribute to the hydrolysis of cAMP in the order PDE-1 > PDE-3 > PDE-4 (Fig. 2A). Hydrolysis of cAMP by all three isozymes was inhibited by incubation of the lysate with 50 µM ZnSO4 30 min before the addition of cAMP. As shown in Fig. 2C, preincubation with zinc ions inhibited PDE-1-mediated cGMP hydrolysis completely, whereas PDE-3 and PDE-4 did not contribute to cGMP degradation. Additionally, the application of all three inhibitors blocked cAMP (Fig. 2B) and cGMP (Fig. 2D) degradation, indicating that there is no noteworthy contribution of other PDE isoforms.



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  		FIGURE 2. Inhibition of cAMP and cGMP hydrolysis by zinc ions in Mono Mac1 lysate. PDE activity was measured by adding cAMP or cGMP (50-µM final concentration) to the lysate of Mono Mac1 cells and monitoring the degradation with HPLC as described in Materials and Methods. Isozyme-selective inhibitors for PDE-1, PDE-3, and PDE-4 were chlorpromazine (170 µM), quazinone (6 µM), and Ro-20-1724 (20 µM), respectively. Values are expressed as the mean ± SEM from at least three independent experiments. Statistically significant (p < 0.05) PDE activity changes compared with control values are indicated (*). A and C, The activities of single PDE isozymes were investigated in the presence of inhibitors for the other two isozymes. To investigate zinc inhibition, lysates were additionally incubated with ZnSO4 (50 µM) 30 min before addition of cAMP (A) or cGMP (C). B and D, Hydrolysis of cAMP (B) and cGMP (D) in untreated lysate compared with PDE activity measured in the presence of inhibitors for PDE-1, PDE-3, and PDE-4.

 
To estimate the concentration of zinc necessary to inhibit PDE, the hydrolysis of cAMP or cGMP was measured in the lysate of Mono Mac1 cells in the presence of different zinc concentrations. As depicted in Fig. 3, zinc inhibited the degradation of both cAMP and cGMP in a dose-dependent manner, with half-maximal inhibition at 8 and 3 µM, respectively.



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  		FIGURE 3. Concentration-dependent effect of zinc on cyclic nucleotide hydrolysis. The lysate of Mono Mac1 cells was incubated for 30 min with zinc concentrations from 1 to 50 µM ZnSO4 before addition of cAMP or cGMP. The degradation of the cyclic nucleotides was measured by HPLC. Results are the mean ± SEM from at least three independent experiments.

 
Reversibility of PDE inhibition by zinc

To study the reversibility of the zinc inhibition of PDE, lysate of Mono Mac1 cells was first incubated with 50 µM ZnSO4, resulting in an inhibition of cAMP and cGMP hydrolysis (Fig. 4A). Upon additional incubation of this lysate for another 1 h with 250 µM of the zinc-specific chelator TPEN, zinc-mediated PDE inactivation was reversed. In contrast, incubation of intact Mono Mac1 cells with 25 µM ZnSO4 plus 50 µM pyrithione for 24 h resulted in decreased cAMP and cGMP hydrolysis that was only partially reversible by addition of TPEN to the lysate (Fig. 4B), indicating a dual effect of zinc on enzyme activity as well as on PDE expression. Therefore, the expression of PDE isozymes was investigated by RT-PCR. After 6 h of zinc treatment, down-regulation of PDE-1C, PDE-4A, and PDE-4B mRNA was observed in Mono Mac1 cells (Fig. 4C). However, zinc had a less pronounced effect on PDE-1B and PDE-4D and did not affect PDE-3B mRNA expression. These latter PDE isozymes might still be expressed and contribute to the remaining PDE activity of ~30%.



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  		FIGURE 4. Effects of zinc ions on PDE activity and mRNA expression. The degradation of cAMP or cGMP was measured by HPLC in the lysate of Mono Mac1 cells (A and B). The values represent the mean ± SEM from at least three independent measurements. Statistically significant (p < 0.05) PDE activity changes are indicated (*). A, Lysate was treated with ZnSO4 (50 µM) for 30 min before addition of cyclic nucleotides (zinc). To assess the reversibility of this incubation, zinc-treated lysate was also incubated with 250 µM of the chelator TPEN for 1 h (TPEN). B, Before lysis, Mono Mac1 cells were incubated for 24 h in the absence or the presence of 25 µM ZnSO4 and 50 µM pyrithione (24h zinc). Lysate of zinc-treated cells was incubated with 250 µM TPEN for 1 h before addition of cyclic nucleotides (TPEN). C, Cells were incubated in the absence or the presence of ZnSO4 (25 µM) plus pyrithione (50 µM) for 6 h. The mRNA expression of PDE subtypes was determined by RT-PCR. Data are representative of three independent experiments.

 
Zinc suppresses LPS-induced up-regulation of PDE-4B expression

In human monocytes the expression of PDE-4B is increased by LPS stimulation (24). Therefore, the role of zinc inhibition in PDE activity and expression was investigated as a possible mechanism to inhibit LPS-induced signals. Treatment of Mono Mac1 cells with LPS for 24 h resulted in up-regulation of PDE-4B mRNA (Fig. 5A) and significantly increased cAMP hydrolysis (Fig. 5B). In accordance with the fact that cGMP is not a substrate for PDE-4, no significant effect of LPS on cGMP degradation was observed (Fig. 5B). Coincubation with zinc and pyrithione eliminated the LPS-induced up-regulation of PDE-4B mRNA expression (Fig. 5A) and reduced the hydrolysis of cAMP and cGMP to levels comparable to those observed with zinc and pyrithione alone (Fig. 5B).



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  		FIGURE 5. Zinc suppresses LPS-induced up-regulation of PDE-4B. Mono Mac1 cells were treated with LPS (250 ng/ml) and ZnSO4 (25 µM) plus pyrithione (50 µM) as indicated. A, PDE-4B mRNA expression measured with RT-PCR after incubation with zinc/pyrithione and LPS for 2 h. As a housekeeping gene, {beta}-actin was used. Data are representative of three independent experiments. B, Total PDE activity in the lysate of Mono Mac 1 cells after 24 h of incubation with LPS and zinc/pyrithione, measured by HPLC using cAMP or cGMP as substrate. The values represent the mean ± SEM from at least three independent experiments. Statistically significant (p < 0.05) PDE activity changes compared with the control values are indicated (*).

 
Down-regulation of LPS-induced release of TNF-{alpha} and IL-1{beta} by zinc ions

Next, the effects of zinc or the unspecific PDE inhibitor, IBMX, on the induction of IL-1{beta} and TNF-{alpha} mRNA expression were investigated in Mono Mac1 cells (Fig. 6A). LPS-induced TNF-{alpha} and IL-1{beta} mRNA expressions were blocked by addition of zinc (25 µM) plus pyrithione (50 µM) as well as by preincubation with IBMX (4 mM). Accordingly, when IL-1{beta} and TNF-{alpha} were measured by ELISA in the supernatant of PBMC, the application of zinc plus pyrithione as well as the unspecific inhibition of PDE by IBMX blocked LPS-induced release of both cytokines (Fig. 6B). To investigate a possible effect of zinc on the secretory level, TNF-{alpha} and IL-1{beta} were measured in supernatant and lysate of primary monocytes after treatment with LPS in the presence or the absence of zinc (Fig. 6C). Zinc abrogated the stimulatory effect of LPS in lysate and supernatant equally, with no zinc-induced accumulation of cytokines in the cellular fraction. Taken together, the data indicate that zinc acts via regulation of the mRNA levels of TNF-{alpha} and IL-1{beta}.



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  		FIGURE 6. Zinc and IBMX down-regulate TNF-{alpha} and IL-1{beta} mRNA and release. Mono Mac1 cells (A) and PBMC (B) were treated with ZnSO4 (25 µM) plus pyrithione (50 µM), LPS (250 ng/ml), and IBMX (4 mM) for 3 h (A) or 24 h (B), respectively. A, TNF-{alpha} and IL-1{beta} mRNA expression were analyzed using RT-PCR, with {beta}-actin as a housekeeping gene. Data are representative of three independent experiments. B, TNF-{alpha} and IL-1{beta} release from PBMC was quantified by ELISA. Values below the lowest standard of 7.8 pg/ml (TNF-{alpha}) or 15.6 pg/ml (IL-1{beta}) were designated not detectable (n.d.). The values represent the mean ± SEM from four different donors. Statistically significant (p < 0.05) changes in TNF-{alpha} and IL-1{beta} concentration compared with cells treated with LPS alone are indicated (*). C, Primary monocytes were stimulated with ZnSO4 (1 µM) plus pyrithione (50 µM) and/or LPS (250 ng/ml) for 24 h. TNF-{alpha} and IL-1{beta} in the supernatant and cellular lysate were determined by ELISA. The values represent the mean ± SEM from three different donors. Statistically significant (p < 0.05) changes in TNF-{alpha} and IL-1{beta} concentration compared with cells treated with LPS alone are indicated (*).

 
Zinc inhibition of PDE elevates intracellular cGMP, but not cAMP

Zinc inhibition of PDE should lead to an elevation of intracellular cyclic nucleotides. However, an increase in cellular cAMP could not be observed when Mono Mac1 cells were treated with zinc (25 µM) and pyrithione (50 µM) for 0.5–6 h (data not shown). Conversely, zinc/pyrithione treatment seemed to have an inhibitory effect on the formation of cAMP, because it significantly reduced the cellular basal level of cAMP and inhibited the increase in cAMP induced by treatment with IBMX together with the AC activator FSK (Fig. 7A). In contrast, incubation of Mono Mac1 cells with zinc plus pyrithione resulted in an elevation of the intracellular cGMP concentration in untreated cells and in the presence of LPS (Fig. 7B). Despite the ability of zinc to inhibit the catalytic activity of all PDE isozymes expressed in Mono Mac1 cells, it increased only the level of cellular cGMP, not that of cAMP.



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  		FIGURE 7. Zinc ions influence the cellular levels of cyclic nucleotides. A, Mono Mac1 cells were stimulated, as indicated, with FSK (10 µM) plus IBMX (4 mM) or ZnSO4 (25 µM) plus pyrithione (50 µM) for 30 min. Cells were lysed, and cAMP concentrations were analyzed as described in Materials and Methods. B, Mono Mac1 cells were treated with ZnSO4 (25 µM) plus pyrithione (50 µM) and LPS (250 ng/ml) for 6 h. Cellular lysates were analyzed by cGMP enzyme immunoassay. The values represent the mean ± SEM from four (A and B) independent experiments, respectively. Statistically significant (p < 0.05) cAMP and cGMP concentration changes are indicated (*).

 
Role of cGMP in zinc inhibition of TNF-{alpha} and IL-1{beta} release

We investigated whether an increase in cellular cGMP can mediate zinc inhibition of LPS-induced cytokine secretion. LPS-induced TNF-{alpha} and IL-1{beta} secretion from PBMC was inhibited by two membrane-permeant cGMP analogues, 8-Br-cGMP and DB-cGMP, in a dose-dependent manner (Fig. 8, A and C). To activate cGMP synthesis by soluble guanylate cyclase (sGC), the NO donor SNOC was used. SNOC administration in the presence of LPS resulted in inhibition of TNF-{alpha} and IL-1{beta} release from human monocytes (Fig. 8, B and D).



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  		FIGURE 8. Role of cGMP in zinc-mediated inhibition of cytokine release. Release of cytokines into the supernatant was quantified by ELISA. Values below the lowest standard of 7.8 pg/ml (TNF-{alpha}) or 15.6 pg/ml (IL-1{beta}) were designated not detectable (n.d.). The values represent the mean ± SEM from at least three different donors. Statistically significant (p < 0.05) concentration changes are indicated (*). A and C, PBMC were stimulated with different concentrations of 8-Br-cGMP or DB-cGMP (30 min before LPS) in the presence of LPS (250 ng/ml). Supernatants were harvested after 24 h and analyzed for TNF-{alpha} (A) and IL-1{beta} (C). B and D, Primary monocytes were treated with 100 or 250 µM SNOC and LPS (250 ng/ml) for 4 h. TNF-{alpha} (B) and IL-1{beta} (C) in the cell culture supernatant were determined by ELISA.

 
The fact that cGMP inhibits LPS-induced TNF-{alpha} and IL-1{beta} release does not necessarily mean that the inhibitory effect of zinc involves cGMP. Therefore, LY83583, an inhibitor of sGC, was used to inhibit cGMP synthesis in primary monocytes. LPS-induced TNF-{alpha} release was not affected by LY83583 (Fig. 9A). However, the effect of 1 µM zinc in the presence of pyrithione, a concentration that suppresses LPS-induced TNF-{alpha} release by >90% in human monocytes, was significantly antagonized by preincubation with LY83583, confirming that cGMP is required for the inhibitory effect of zinc on TNF-{alpha} release. In contrast to TNF-{alpha}, LPS-induced release of IL-1{beta} was increased in the presence of LY83583. Because the concentration of cGMP did not increase during stimulation with LPS (Fig. 7B), basal levels of cellular cGMP seem to inhibit the release of this cytokine. Nevertheless, zinc-mediated suppression of LPS-induced IL-1{beta} release was also reversed by LY83583 (Fig. 9B), confirming a role for cGMP in the effect of zinc on both cytokines.



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  		FIGURE 9. Reversal of zinc-mediated TNF-{alpha} and IL-1{beta} suppression by inhibition of sGC. Monocytes were stimulated with LPS (250 ng/ml), zinc (1 µM) plus pyrithione (50 µM), and 1 µM LY83583 (added 30 min before LPS and zinc/pyrithione) as indicated. The TNF-{alpha} (A) and IL-1{beta} (B) contents of the supernatant were analyzed by ELISA after 4 h of stimulation. Values below the lowest standard of 7.8 pg/ml (TNF-{alpha}) or 15.6 pg/ml (IL-1{beta}) were designated not detectable (n.d.). The values represent the mean ± SEM from three different donors, with measurements performed in duplicate. Statistically significant (p < 0.05) concentration changes are indicated (*).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Zinc supplementation has seemingly contradictory effects on LPS-induced production of proinflammatory cytokines. It stimulates the release of the monokines IL-6, IL-1{beta}, and TNF-{alpha} in human PBMC (2); induces TNF-{alpha} mRNA expression (3); and acts synergistically with LPS if applied simultaneously in substimulatory concentrations (5). In contrast, zinc was found to negatively regulate TNF-{alpha}, IL-8, and IL-1{beta} gene expression in the HL-60 monocyte/macrophage cell line (4), and zinc pretreatment attenuates TNF-{alpha} production during liver injury in mice, thereby protecting against LPS-induced hepatotoxicity (6). Fig. 1 shows that these effects are not mutually exclusive, but occur in the same experimental system depending on the concentration of zinc. Low concentrations have a synergistic effect together with LPS and are proinflammatory, whereas higher concentrations are anti-inflammatory and inhibit monokine production. The stimulatory effects of zinc may be explained by the ability of zinc ions to enhance the effects of LPS by modulating its fluidity state (25) and more generally by their capability to activate a multitude of different signal transduction pathways, especially protein phosphorylation (26).

In this report the mechanisms that lead to the inhibitory effect of zinc were investigated. Zinc has been reported to increase cGMP levels in PC12 cells by inhibition of PDE activity (17). Agonists of both cAMP and cGMP suppress LPS-induced production of TNF-{alpha} (8, 12). Additionally, PDE-4-specific inhibitors, such as rolipram and Ro-20-1724, attenuate TNF-{alpha} secretion from PBMC and monocytes by elevation of intracellular cAMP (7, 8, 9, 10, 11). Thus, we investigated whether zinc interferes with monokine production via modulation of cyclic nucleotide signaling. A dual effect of zinc on PDE activity was found in Mono Mac1 cells. First, the enzymatic activity of PDE is directly and reversibly inhibited by zinc ions. Second, zinc inhibits the gene expression of several PDE subtypes and can also block the LPS-stimulated increase in PDE-4B expression. To date, PDE-1, PDE-3, and PDE-4 have been reported to be expressed in monocytes, and the use of isozyme-selective inhibitors confirms that PDE activity in Mono Mac1 lysate is mediated solely by these three isozymes. All isoforms were inhibited by zinc, which blocked the degradation of both cGMP and cAMP in Mono Mac1 lysate. In contrast, only the level of cellular cGMP increased after incubation of whole Mono Mac1 cells with zinc, whereas cAMP was slightly reduced, and zinc even inhibited the increase in cAMP induced by treatment with IBMX and FSK. A possible explanation is an inhibitory effect of zinc on cAMP synthesis, because it has been reported that zinc inhibits the GTPase activity from the stimulatory GTP-binding protein, a regulator of AC, and thereby abrogates the stimulation of AC activity (27). This would render the inhibition of cAMP-degrading PDE ineffective due to a lack of cAMP formation. It is unknown whether zinc has a similar effect on the receptor-associated GC, but cGMP is also produced by sGC, which is stimulated by NO, but not by G proteins. An involvement of sGC in the zinc-mediated suppression of TNF-{alpha} and IL-1{beta} was confirmed by the reversal of this suppression with LY83583 (Fig. 9), a competitive inhibitor of sGC (28). The increase in LPS-induced secretion of IL-1{beta}, but not TNF-{alpha}, by LY83583 in the absence of zinc may be explained by the different proteases involved in processing of the precursor proteins. Although TNF-{alpha} is cleaved by the metalloprotease ADAM-17, IL-1{beta} processing and activation involves the IL-1-converting enzyme, a caspase family cysteine protease. An inhibition of caspase activity through cGMP-dependent pathways has been demonstrated previously (29). When the NO donor SNOC was used to activate sGC, LPS-induced TNF-{alpha} and IL-1{beta} release was suppressed. However, NO also releases protein-bound zinc by oxidation of zinc-binding cysteine residues (30), and cytokine-induced NO production is sufficient to cause measurable intracellular zinc release (31). Such an increase in available zinc may inhibit cyclic nucleotide degradation via PDE inhibition and act synergistically with the activation of sGC. Because the intracellular concentration of available zinc fluctuates (26), it can be speculated that these fluctuations might influence PDE activity and, thus, cyclic nucleotide signaling. Similar to PDE, zinc inhibits protein tyrosine phosphatases at low micromolar concentrations in cellular lysate (32). An inhibition of these phosphatases by cellular zinc has been shown to participate in the regulation of insulin and insulin-like growth factor-I-modulated tyrosine phosphorylation (32), and it would be interesting to determine whether PDE are affected by cellular zinc in a similar fashion. Plasma contains 12–16 µM zinc (1), and the average concentration of zinc in tissues such as skeletal muscle, kidney, and liver is several hundred micromolar (33). Zinc even reaches millimolar concentrations in zinc-rich tissue such as prostate (34). At these concentrations, PDE should be permanently inactive. Nevertheless, most of that zinc is tightly bound to proteins, and the amount available to inhibit PDE is much lower, but zinc can easily reach concentrations that block TNF-{alpha} release in cultured cells and in vivo (4, 6).

Zinc does not inhibit TNF-{alpha} and IL-1{beta} secretion, but, rather, affects cytokine mRNA levels. In what way a rise in cGMP influences the signal transduction that leads to the transcription of TNF-{alpha} and IL-1{beta} remains to be clarified. The major cGMP target in many cell types is protein kinase G, and cGMP-dependent inhibition of Raf-1 by phosphorylation on serine 43 by protein kinase G has been reported (35). High cGMP concentrations can cross-activate protein kinase A and lead to ERK as well as Raf-1 inhibition (36, 37). Also, destabilization of TNF-{alpha} mRNA by cGMP in LPS-activated macrophages was discussed previously (38). The inhibition of TNF gene expression by atrial natriuretic peptide has been found to be a cGMP-mediated effect, involving inhibition of AP-1 and NF-{kappa}B activation (12, 39). Which of these mechanisms contribute to zinc inhibition of LPS-induced cytokine production, and the possible involvement of other signaling pathways, are currently under investigation.

Under the experimental conditions used in this study, zinc exerts short-term anti-inflammatory effects via cGMP signals. Down-regulation of PDE-4 expression shows the ability of zinc to act as a modulator of cAMP metabolism as well. PDE-4 is particularly abundant in cells of the immune system, and its inhibitors are investigated as potential anti-inflammatory and immunomodulatory agents to inhibit the synthesis of proinflammatory cytokines and reactive oxygen species. Potential therapeutic targets for PDE-4 inhibition include not only respiratory diseases such as asthma and chronic obstructive pulmonary disease, but also multiple sclerosis, rheumatoid arthritis (RA), diabetes, and septic shock (40, 41). Many of these diseases, such as asthma (42), chronic obstructive pulmonary disease (43), RA (44), and diabetes (45) are associated with reduced serum zinc levels. Elderly individuals, who generally tend toward reduced serum zinc levels, have a significantly increased constitutive production of IL-1, IL-6, and IL-8 as well as an elevated release of these cytokines after stimulation of whole blood samples with LPS compared with young donors (46). Serum levels of TNF-{alpha} and IL-1{beta} correlate negatively with the zinc status in RA patients (44). These two cytokines are also potential candidates for {beta}-cell damage in type 1 diabetes mellitus (45), and zinc supplementation prevents spontaneous and streptozotocin-induced diabetes in NOD mice (47). Therapeutic administration of zinc or, more generally, a correction of a disturbed zinc homeostasis to restore the plasma zinc levels to normal may be ways to modulate cyclic nucleotide levels and thus achieve beneficial results in several inflammatory diseases.

The low acute toxicity of zinc indicates that zinc supplementation could be a potential therapy with few side effects. However, the doses, manners, and times of administration of zinc will have to be carefully taken into consideration, because the concentration-dependent pro- or anti-inflammatory effects harbor potentially harmful consequences. This is best demonstrated by the effects of zinc administration during septic shock. Within a narrow concentration range, zinc exerts protective effects in endotoxin-challenged mice (48). Although pretreatment with zinc protects against mortality, simultaneous administration of zinc and LPS increases TNF-{alpha} serum levels, resulting in severe proinflammatory action of zinc and enhanced mortality (48, 49, 50). Zinc has the potential to be an immune modulatory agent, either through the stimulatory effect of zinc on monocytes or by PDE-mediated suppression of proinflammatory cytokine release in autoimmune disease and septic shock. However, only a thorough understanding of zinc metabolism far beyond our present knowledge, including its uptake and distribution as well as its effects on cellular and molecular levels, will allow us to exploit the full immunomodulatory potential of zinc.


   Acknowledgments
 
We thank Dr. Marcus V. Cronauer (Heinrich Heine University, Dusseldorf, Germany) for human prostate cDNA, and Romney Haylett for critical reading of the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by the START program of the medical faculty, RWTH Aachen University, and Grant Ha 4318/3-2 from Deutsche Forschungsgemeinschaft. Back

2 Address correspondence and reprint requests to Dr. Hajo Haase, Institute of Immunology, University Hospital, RWTH Aachen University, Pauwelsstrasse 30, 52074 Aachen, Germany. E-mail address: hhaase{at}ukaachen.de Back

3 Abbreviations used in this paper: AC, adenylate cyclase; 8-Br-cGMP, 8-bromo-cGMP; DB-cGMP, dibutyryl cGMP; FSK, forskolin; IBMX, 3-isobutyl-1-methylxanthine; PDE, phosphodiesterase; RA, rheumatoid arthritis; sGC, soluble guanylate cyclase; SNOC, S-nitrosocysteine; TPEN, N,N,N',N',-tetrakis (2-pyridyl-methyl)-ethylenediamine. Back

Received for publication March 23, 2005. Accepted for publication July 22, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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