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The role of zinc in calprotectin expression in human myeloid cells
Journal of Trace Elements in Medicine and Biology 49 (2018) 106–112
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The role of zinc in calprotectin expression in human myeloid cells Simone Lienau, Lothar Rink, Inga Wessels
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Institute of Immunology, Medical Faculty, RWTH Aachen University, Pauwelsstr. 30, D-52074, Aachen, Germany
A R T I C LE I N FO
A B S T R A C T
Keywords: Zinc deficiency Calprotectin Myeloid cells Monocytic cells Inflammation Inflammatory disease
Elevated levels of calprotectin and other inflammatory mediators have been observed in inflammatory diseases paralleling serum hypozincemia. While a role of zinc in the regulation of tumor necrosis factor α, interleukin (IL)-1β and IL-6 expression has been established, the direct interrelation of zinc and calprotectin (S100A8/ S100A9 heterodimer) expression is so far missing. In the present study, we analyzed mRNA and protein levels of S100A8 and S100A9 in monocytic Mono Mac (MM)1 and early myeloid THP-1 and U937 cells to elucidate the effect of zinc deficiency on their expression. We could depict that zinc deficiency alone enhances mRNA and protein expression of calprotectin in myeloid cells, independently from maturity stage. Moreover, pre-existing zinc deficiency augmented lipopolysaccharide (LPS)-induced calprotectin expression in CD14+ MM1, but not in CD14− U937 or CD14− THP-1 cells. Zinc deficiency and LPS seem therefore to activate different intracellular pathways. Our findings suggest that zinc does not only regulate the activity of calprotectin but also its expression by human myeloid cells.
1. Introduction
detail so far, but is important to understand to develop new treatment strategies for these diseases. Hence, the aim of this study was to investigate the influence of zinc homeostasis on calprotectin expression in human myeloid cells of different maturity stages.
A balanced zinc homeostasis is crucial for an appropriate immune response [1]. Zinc deficiency caused by infections, malnutrition, age or diseases may therefore lead to complex immune impairments [2–5]. In the elderly, zinc deficiency was associated with constantly elevated inflammatory parameters [6] and increasing rates of infectious diseases with high mortality therefrom [5,7]. Generally, severe and chronic serum hypozincemia are accompanied by an increase of pro-inflammatory mediators like tumor necrosis factor α, interleukin (IL)-1β, IL-6 and calprotectin [5,8–10]. Calprotectin is a heterodimeric complex belonging to the S100 family, consisting of the subunits S100A8 and S100A9. It can influence zinc metabolism by chelating zinc in the presence of calcium. Amongst other roles, it thereby deprives bacteria of zinc, conveying antimicrobial properties [11]. Predominantly it is expressed by cells of the innate immune system including neutrophil granulocytes and monocytes [12]. Hallmarks of inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease, cystic fibrosis or sepsis are elevated levels of calprotectin in serum paralleled by serum hypozincemia [8,9,13,14]. A prior report links zinc deficiency to the upregulation of calprotectin [15]. The precise association of zinc with calprotectin expression especially in an inflammatory context has not been investigated in
2. Methods and materials 2.1. Cell culture MM1, THP-1 and U937 cells were cultivated at 37 °C, humidified atmosphere and 5% CO2. Culture medium for U937 was RPMI 1640 (Sigma-Aldrich, München, Germany) with 10% heat-inactivated, lowendotoxin FCS (PAA, Cölbe, Germany), 100 U/ml penicillin (SigmaAldrich), 100 μg/ml streptomycin (Sigma-Aldrich) and 2 mM L-glutamine (Sigma-Aldrich). MM1 cells were cultured in U937 medium where 1% non-essentialamino acids (NEAA, Lonza, Köln, Germany) and 1% sodium pyruvate (Lonza) were added. To culture THP-1 cells, 2.5 μl β-Mercaptoethanol (Merck, Darmstadt, Germany) were added per 500 ml of U937 medium. For zinc-deficient medium, zinc adequate control medium with 10% FCS was treated with CHELEX 100 ion exchange resin (Sigma-Aldrich) for 1 h at 20 °C and was subsequently reconstituted with 500 μM CaCl2
Abbreviations: AAS, atomic absorption spectrometry; IL, interleukin; LPS, lipopolysaccharide; MT, metallothionein; MM1, Mono Mac 1; TNF, tumor necrosis factor; TPEN, N,N,N′,N′tetrakis(2-pyridylmethyl)ethylenediamine ⁎ Corresponding author. E-mail address: [email protected] (I. Wessels). https://doi.org/10.1016/j.jtemb.2018.04.022 Received 12 January 2018; Received in revised form 2 April 2018; Accepted 19 April 2018 0946-672X/ © 2018 Elsevier GmbH. All rights reserved.
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(Sigma-Aldrich) and 400 μM MgCl2 (Merck). The metal composition of RPMI 1640 medium with 10% FCS prior and after treatment with CHELEX resin can be found in Mayer et al. [16]. The cells were pre-cultivated for 24 h either in zinc adequate control medium, in zinc deficient medium (CHELEX) as described above, or in zinc reconstituted CHELEX-medium (CHELEX + Zn) with ZnSO4 (ZnSO4 × 7 H2O, Merck) to yield a final concentration of 8 μM. Subsequently lipopolysaccharide (LPS), E. coli serotype O111:B4 (Sigma-Aldrich) was added for another 24 h treatment as indicated in the figure legends.
SYBR Green PCR Master Mix (Applied Biosystems). Fold-changes were calculated through the ΔΔ CT method. 2.7. Cell extracts and Western blotting A total of 1 × 106 cells were lysed in 100 μl lysis buffer (0.5 M Tris–HCl (pH 6.8) (Carl Roth, Karlsruhe, Germany), 2% (w/v) sodium dodecyl sulfate (Merck), 1 mM sodium vanadate (Sigma-Aldrich), 26.6% glycerol (Carl Roth), 1% (v/v) β-mercaptoethanol, 0.01% (w/v) bromophenol blue (Fluka, Buchs, Switzerland)). The cells were sonicated for 15 s and then boiled for 3 min at 95 °C. An equivalent of 2 × 105 cells per lane was separated on 15% polyacrylamide (S100A9 and S100A8) or 10% polyacrylamide gels (βactin) at 150 V and blotted to nitrocellulose membranes. Homogeneous loading of membranes was confirmed with Ponceau S staining (SigmaAldrich). Following destaining, membranes were blocked with tris buffered saline (TBS, 20 mM Tris-HCl (pH 7.6, Carl Roth), 136 mM NaCl (Merck)) including 0.1% (v/v) Tween 20 (AppliChem, Darmstadt, Germany)) and 3% bovine serum albumin (Fluka) and incubated overnight with the primary antibodies against S100A9, S100A8 (Santa Cruz Biotechnology, Heidelberg, Germany) or β-actin (Cell Signaling Technology, Frankfurt am Main, Germany) at 1:200 dilutions in TBS-T containing 5% bovine serum albumin. Membranes were then incubated with horseradish peroxidase (HRP)-linked anti-rabbit IgG secondary antibody and HRP-coupled anti-biotin antibody for detection of biotinlabeled molecular weight (MW) standard (anti-biotin 1:1000, antirabbit 1:2000; Cell Signaling Technology) for 2 h. This was followed by detection with LumiGlo reagent (Cell Signaling Technology) on a LAS3000 (Fujifilm Lifescience, Düsseldorf, Germany).
2.2. Measurement of free intracellular zinc with ZinPyr-1 Free intracellular zinc was measured using ZinPyr-1 (10 μM, Santa Cruz Biotechnology, Heidelberg, Germany). The mean fluorescence signal was detected by FACSCalibur (BD Biosciences, Heidelberg, Germany) using Cellquest software 3.0. The formula [Zn] = KD× [(F − Fmin)/(Fmax − F)] was used to calculate the concentration of free labile intracellular zinc. In doing so, the dissociation constant (KD) was 0.7 nM for the Zn/ZinPyr-1 complex [17]. The maximal (Fmax) and minimal (Fmin) mean fluorescence signals were generated by addition of zinc (100 μM) combined with pyrithione (50 μM) (Sigma-Aldrich) or N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (50 μM) (TPEN, Sigma-Aldrich), respectively [18]. 2.3. ELISA Supernatants were harvested after stimulation, stored at −20 °C until measurement, and only thawed once for cytokine detection. IL-6 protein production was quantified using OptEIA assays (BD Biosciences) according to the manufacturer’s instructions.
2.8. Statistical analysis Statistical significance of the results was analyzed by GraphPad Prism software version 5 (GraphPad software, La Jolla, CA, USA). In the case of Gaussian distribution, as tested by D’Agestino’s K-square test of normality, student’s t-test for unpaired samples was performed. Without this assumption, Mann-Whitney-U test was performed for unpaired samples and Wilcoxon signed rank test was performed for paired samples. p-values ≤ 0.05 were regarded statistically significant and are illustrated in the figures by *, p-values ≤ 0.01 are shown by ** and pvalues ≤ 0.001 are shown by ***.
2.4. Flow cytometry Cells were stained with phycoerythrin (PE)-conjugated CD14 monoclonal antibody (mAbs; BD Biosciences) or with the isotypic control PE-conjugated immunoglobulin IgG2bκ mAb (BD Biosciences) for 15 min. Washed cells were then analyzed in a FACSCalibur (BD Biosciences) using Cellquest software 3.0. 2.5. Atomic absorption spectrometry (AAS)
3. Results Cells were washed with washing buffer (0.9% sodium chloride (NaCl, Merck), 10 mM ethylenediaminetetraacetic acid (EDTA, Merck) and 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES, AppliChem, Darmstadt, Germany)) twice and scraped into icecold water. Subsequently, cells were hydrolyzed in 200 μl HNO3 (> 69.5%, for trace analyses, Sigma-Aldrich) for 2 h at 80 °C. Cellular zinc concentration was measured by flame atomic absorption spectrometry (AAS, Perkin Elmer, Baesweiler, Germany) using a standard curve made from AAS grade zinc standard solution. Absorbance values were normalized to cell counts.
3.1. Zinc homeostasis of MM1 cells during zinc deficiency and after LPSstimulation Initially, we confirmed that pre-incubation in CHELEX-treated medium decreased intracellular free zinc levels in MM1 cells (Fig. 1A), to verify our zinc deficiency model [15,16]. Intracellular free zinc was decreased significantly in MM1 cells by 58.2% to a mean of 0.028 nM (Fig. 1A) incubated in CHELEX-treated medium compared with the zinc adequate controls. However, intracellular zinc levels of zinc adequate controls and zinc deficient MM1 cells were not affected significantly by LPS stimulation for 24 h, compared with the matching unstimulated control. In order to eliminate artefacts from CHELEX resins, we used a second control sample, which comprised of CHELEX-treated medium, replenished with zinc to yield a final concentration of 8 μ M zinc, comparable to regular zinc adequate medium. No significant difference was detected between MM1 cells incubated in regular zinc adequate control medium or in zinc replenished CHELEX-treated medium (Fig. 1B). Due to simplification, only one control group, i.e. cells cultured in regular medium, was used consequentially during further experiments. To support our data for the intracellular free zinc measurements, we used atomic absorption spectrometry to determine total zinc levels in
2.6. Reverse transcription and real-time PCR RNA was isolated using the NucleoSpin® RNA Kit (Macherey-Nagel, Düren, Germany) and cDNA was acquired through use of qScriptTM cDNA Synthesis Kit (Quanta Biosciences, Darmstadt, Germany) according to the manufacturers´ protocols. The primers for S100A8 and S100A9 [15] and the primers for the housekeeping gene glycerinaldehyde 3-phosphate dehydrogenase (GAPDH) [19] were used as previously described. All samples were run using a Step-1 plus (Applied Biosystems, Darmstadt, Germany) with the following parameters: 95 °C for 15 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min for S100A8 and GAPDH and 61 °C for S100A9 in duplicate, using Power 107
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Fig. 1. Zinc homeostasis of myeloid cells during zinc deficiency and after LPS-stimulation. (A) MM1 cells were either pre-incubated in zinc deficient medium (CHE), or in zinc adequate control medium for 24 h with or without subsequent LPS (250 ng/ml) stimulation for 24 h. Intracellular free zinc was detected by flow cytometry using the fluorescent zinc probe ZinPyr-1. Charted are mean values ± SD from n = 5 independent experiments. Statistical significances, quantified by Mann-Whitney U test, are illustrated (*, p ≤ 0.5). (B) The same experiment as in (A) was performed for MM1 comparing the values for the zinc adequate control medium with the values for CHELEXtreated medium, where zinc was replenished to yield a final concentration of 8 μ M. Cells were additionally stimulated with LPS as indicated (250 ng/ml, 24 h). All values are shown as mean ± SD from n = 5 independent experiments. No significant differences have been detected between the two comparable pairs by Mann-Whitney U test. (C, D) Total cellular zinc levels of MM1 cells (C) or their supernatants (D) cultured as in (A) were measured via atomic absorption spectrometry. The bars represent mean values ± SD from (C) n = 9 and (D) n = 4 independent experiments. Statistical significances, calculated by Mann-Whitney U test, are depicted (*, p ≤ 0.5; **, p ≤ 0.01; ***, p ≤ 0.001).
3.3. Zinc deficiency and LPS stimulation cumulatively activate calprotectin protein expression in MM1 cells
MM1 cells. Compared with the control, total cellular zinc levels were significantly decreased by 20.6% to a mean of 0.116 mg/103 cells in cells cultured in CHELEX-treated medium (Fig. 1C). Cells cultured in CHELEX-treated medium may thus further on be called zinc deficient. In contrast to intracellular free zinc levels, total cellular zinc as determined via AAS analysis was significantly increased by LPS stimulation by 21.9% to a mean of 0.178 mg/103 cells (Fig. 1C). In accordance with these findings, zinc levels in the supernatant were significantly decreased in case of LPS stimulation in contrast to the untreated control (Fig. 1D) and by 33.3% to a mean of 0.002 mg/103 cells in case of LPS stimulation after pre-incubation in zinc deficiency in contrast with the corresponding control. Our findings confirmed that incubation in zinc deficient medium decreases free intracellular as well as total cellular zinc in myeloid cells.
Subsequently, we explored calprotectin protein expression in MM1 cells using the same experimental settings. Western blotting (Fig. 2C) and densitometry (Fig. 2D, E) analyses revealed, similar to what was observed for mRNA expression, that zinc deficiency alone significantly increased S100A9 protein levels more than two fold-change (S100A9/ β-actin) compared with the zinc adequate controls (Fig. 2E). Further, LPS stimulation alone significantly activated S100A9 protein expression doubling it as well, which was then significantly further augmented by pre-existing zinc deficiency (Fig. 2E). Trends for S100A8 protein expression were similar (Fig. 2D), but did not reach significance, although elevation of mRNA levels was observed (Fig. 2A). Taken together, our data shows increased calprotectin expression in zinc deprived circumstances in MM1 cells.
3.2. Calprotectin mRNA expression by MM1 cells is up-regulated by zinc deficiency and LPS stimulation
3.4. CD 14 expression and LPS-induced cytokine production is limited to MM1 cells
An association between zinc homeostasis and calprotectin expression has been suggested, but was not investigated in detail so far. Thus, we examined the effects of zinc deficiency on the regulation of calprotectin mRNA expression, with or without the presence of LPS stimulation to model an infection, by real-time PCR. After cultivation in zinc deficient medium, the expression of S100A8 and S100A9 mRNA in MM1 cells was significantly elevated reaching over 5 fold (S100A8 mRNA/GAPDH) and 3 fold (S100A9 mRNA/GAPDH) the expression of zinc adequate controls (Fig. 2A, B). Furthermore, stimulation of zinc adequate controls with LPS for 24 h significantly activated S100A8 and S100A9 mRNA expression by 998% to a mean fold-change of 10.98 (S100A8 mRNA/GAPDH) and by 679.6% to a mean fold-change of 7.796 (S100A9 mRNA/GAPDH). Zinc deficiency preceding LPS stimulation significantly boosted LPS-induced mRNA expression of S100A8 and S100A9 in MM1 cells, compared to zinc deficiency or LPS stimulation alone. These data revealed that zinc deficiency leads to a rise of calprotectin mRNA expression in monocytic MM1 cells and augments its expression during inflammatory settings.
Results from analyses in MM1 cells showed a synergistic effect of zinc deficiency and LPS stimulation on the expression of calprotectin. This suggests that independent pathways may be activated here by zinc deficiency compared to LPS stimulation. In order to distinguish effects mediated via CD14-dependent pathways from processes mediated via alternative pathways, we examined THP-1 and U937 cells, which do not express significant amounts of CD14 on their surface as verified by flowcytometric investigation and compared results to those from MM1 cells, positive for CD14 expression (Fig. 3A–C). In addition, only MM1 cells were able to secrete significant amounts of IL-6 after LPS stimulation, whereas no significant production was detected for THP-1 and U937 cells (Fig. 3D) confirming that CD14-dependent signaling pathways are not activated by LPS in U937 and THP-1. This rendered THP-1 and U937 ideal for investigating CD14-independent pathways. Investigation of alternative cell lines also enabled us to generate a more representative model for human myeloid cells, to eliminate cell line specific effects, and effects elicited by the 108
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Fig. 2. Zinc-homeostasis is involved in the regulation of calprotectin mRNA and protein expression in myeloid cell lines. MM1 cells were pre-incubated in zinc adequate control medium or zinc deficient medium (CHE) for 24 h and subsequently stimulated with LPS (250 ng/ml, 24 h). (A, B) mRNA from MM1 cells was collected, reverse transcribed and quantified by real-time PCR using SBYR-Green. The gene expression of S100A8 (A) and S100A9 (B) was normalized to the housekeeping gene GAPDH. The fold changes were calculated by the ΔΔ CT-method for n = 7 independent experiments. Shown are mean values ± SD. Statistically significant differences are marked (*, p ≤ 0.5; **, p ≤ 0.01) as quantified by Mann-Whitney-U test. (C, D, E) Human calprotectin protein expression in MM1 cells was analyzed by western blotting with antibodies against S100A8, S100A9 and β-actin (C). The relative density of the protein bands of S100A8 (D) and of S100A9 (E) was quantified with regard to the control β-actin. For (D) n = 4 and for (E) n = 13 independent experiments were performed and values are shown as mean ± SD. Statistically significant differences are marked (*, p ≤ 0.5; **, p ≤ 0.01; ***, p ≤ 0.001) as quantified by Mann-Whitney-U test.
degeneration of a certain tumor cell line.
3.6. Regulation of calprotectin mRNA expression in THP-1 and U937 cells by zinc deficiency
3.5. Zinc homeostasis of THP-1 and U937 cells during zinc deficiency and after LPS-stimulation resembles findings for MM1 cells
Subsequently, we examined the expression of S100A8 and S100A9 mRNA in THP-1 and U937 cells. Contrasting the findings in MM1 cells (Fig. 2A, B), LPS stimulation alone was not able to significantly induce S100A8 and S100A9 mRNA expression in both THP-1 (Fig. 5A, B) and in U937 (Fig. 5C, D) cells compared with the unstimulated control. However, a rise in the mRNA expression of S100A8 and S100A9 after cultivation in a zinc deficient environment was observed in comparison with the control (Fig. 5A–D). This was significantly increased for S100A8 mRNA of THP-1 cells. Hence, our results demonstrate that zinc deficiency increases the expression of calprotectin in all examined human myeloid cell lines.
Experiments with the rather immature myeloid cell lines THP-1 and U937 were done in a uniform manner as for the MM1 cells. Fig. 4 illustrates our findings that the intracellular zinc content in THP-1 (Fig. 4A) and in U937 (Fig. 4B) was significantly decreased by 57.3% to a mean zinc concentration of 0.053 nM and 41.9% to a mean of 0.036 nM respectively in cells cultured in zinc deficient medium for 24 h in comparison with the respective control. No significant differences in the intracellular zinc content could be demonstrated for these cell lines after LPS stimulation for further 24 h in comparison with the matching control. This is in accordance with our findings for MM1 cells.
4. Discussion This present study provides evidence for a regulatory role of zinc homeostasis not only in calprotectin activity, but also in its expression 109
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Fig. 3. Analysis of CD 14 expression on the surface of human cell lines as a marker for myeloid differentiation. Flow cytometry was used to detect the expression of CD14 on the surface of MM1 (A), THP-1 (B) and U937 cells (C). Shown is one representative example of three individual experiments, respectively. The graphs show the counts of CD14 positive cells in comparison to the matching isotypic control phycoerythrin-conjugated immunoglobulin IgG2bκ mAb. Furthermore, zinc adequate U937, THP-1 and MM1 cells were stimulated with LPS (100 ng/ml, 24 h) and IL-6 secretion was analyzed in their supernatants. (D) presents n = 6 independent ELISA’s and values are shown as mean ± SD. Statistical significances, calculated by Mann-Whitney U test, are depicted (*, p ≤ 0.5).
LPS stimulation for 24 h did not show a long-term effect on the measured intracellular zinc levels in these cells. Previous studies by Haase et al. revealed an instant increase of free intracellular zinc within minutes after treatment with LPS. They could show that nearly normal zinc levels were recovered in less than one hour post stimulation [24]. Our time of measurement was 24 h after the stimulation with LPS. This suggests that the short-term effect of a LPS-induced increase of free zinc, a so-called zinc flux, has already been normalized at the time of our measurement, explaining why no effects of LPS on intracellular free zinc levels were observed here. AAS measurements of total cellular zinc confirmed the decrease of the intracellular free zinc levels in MM1 cells after incubation in zinc deficient medium. This indicates that in response to zinc deficient conditions, myeloid cells do loose the trace element and do not only redistribute it into cell organelles or attach it to zinc binding proteins like metallothionein in case of zinc deficiency. In contrast, stimulation with LPS led to a significant rise in total cellular zinc and to a corresponding significant drop of total zinc in the supernatant. One can hypothesize that zinc is taken up by the cell, shortly after LPS stimulation, as suggested by Haase et al. [26]. Subsequently, zinc is probably stored in compartments or tightly bound to zinc-binding proteins, rendering it undetectable if free zinc is
in human myeloid cell lines. More precisely, zinc deficiency alone was shown to induce calprotectin expression in our experimental setting and to augment LPS-induced calprotectin expression. Zinc deficiency may be classified depending on its severity. Slight zinc deficiency can be regularly found in the elderly causing mild immune disturbances [20]. Severe zinc deficiency was correlated with overshooting immune responses and elevated levels of calprotectin in sepsis patients and associated with sepsis initiation and progression in these patients [21]. Although intracellular free zinc levels only represent a small proportion of the total cellular zinc content [22], free zinc is essential for a plurality of cellular processes like intracellular communication [16], second messenger function [23] or regulation of cytokine production [24]. The free intracellular zinc pool is quickly exchangeable, due to the possibility to release zinc promptly from metal binding proteins like metallothionein (MT) [25]. Like MT, Calprotectin is also able to sequester zinc and is expressed mainly by granulocytes and monocytes [12]. Intracellular free zinc concentrations were measured around 0.07 nM in MM1 cells, consistent with former findings [24]. While we could reproduce a decrease of intracellular zinc in the investigated cell lines after pre-incubation in zinc deficient medium [15,16], subsequent
Fig. 4. Zinc homeostasis of THP-1 and U937 during zinc deficiency and after LPS-stimulation. THP-1 (A) and U937 (B) cells were either pre-incubated in zinc deficient (CHE), zinc reconstituted CHELEX-medium (CHE + Zn) or zinc adequate control medium for 24 h and stimulated with LPS (250 ng/ml) for additional 24 h. Intracellular free zinc was detected by flow cytometry using the fluorescent zinc probe ZinPyr-1. Charted are mean values ± SD from (A) n = 5 and (B) n = 3–6 independent experiments. Statistical significances, quantified by Mann-Whitney U test, are illustrated (*, p ≤ 0.5; **, p ≤ 0.01). 110
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Fig. 5. Zinc deficiency upregulates calprotectin mRNA expression in THP-1 and U937 cells. Same experiments as in (2A and B) were performed for THP-1 (5A, B) and for U937 (5C, D) cells. In (A) the expression of S100A8 mRNA in THP-1 cells normalized to the housekeeping gene GAPDH is shown, whereas in (5B) the expression of S100A9 mRNA in THP-1 cells is presented. Consistent, the same experiments were implemented for U937. Depicting in (C) is the mRNA expression of S100A8 and in (D) the expression of S100A9 mRNA. Data are shown as mean from n = 9 (A, B) and n = 3 (C, D) independent experiments ± SD. Statistically significant differences, quantified by MannWhitney-test, are marked (*, p ≤ 0.5; **, p ≤ 0.01).
Moreover, our data are in line with the previous findings of Mazzatti et al. They showed that calprotectin subunits S100A8 and S100A9 were upregulated in myeloid THP-1 cells after cultivation with the zinc chelator TPEN (N,N,N',N'-tetrakis(2-pyridyl-methyl)ethylenediamine) for 40 h [15]. We extended the experimental settings by investigating three different myeloid cell lines of different maturity stages and using CHELEX resin instead of TPEN to generate models for pre-existing zinc deficiency. One disadvantage of TPEN is that it was shown to effect cell metabolism directly in addition to effects via its zinc chelating capacity. Additionally, one major benefit of CHELEX resins is that they withdraw zinc from medium with over 90% efficiency and that resins are separated from the medium before the latter is used for cell culture [16]. While Mazzatti et al. [15] investigated only the effect of zinc deficiency, we additionally examined the effect of zinc deficiency in an inflammatory setting and stimulated the cells with LPS for 24 h. Dubben et al. found out that during 1α,25-dihydroxyitamin D3-induced differentiation, the intracellular free zinc levels in the monocytic cell line HL-60 declined while the expression of S100A8 and S100A9 increased [32]. This underlines our finding that calprotectin expression increases with decreasing intracellular zinc levels. Our results from calprotectin mRNA expression analyses in MM1 suggested that independent pathways may be activated by zinc deficiency compared to LPS stimulation. LPS-mediated signaling is well defined and requires mainly activation via CD14 [33]. The surface marker CD14 is expressed on monocytes and further activates the tolllike receptor (TLR)-4 in those cells after binding to LPS. Subsequently it can activate a signal cascade via the nuclear factor kappa-light-chainenhancer of activated B cells (NF-κB), which among others is liable for the secretion of pro-inflammatory cytokines like IL-1β, IL-6 and TNF-α [24,25,34]. However, it has been shown that expression of some proinflammatory mediators cannot be completely blocked by CD14-specific monoclonal antibodies [35], suggesting alternative pathways. In order to discern effects mediated via CD14-induced signaling pathways from other cellular mechanisms, we examined CD14+ MM1 compared to CD14− and LPS-unresponsive THP-1 and U937 cells. CD14 appears to be necessary to enable LPS-induced calprotectin expression. However, CD14− U937 and THP-1 cells were still able to express increased amounts of calprotectin mRNA during zinc deficiency. This
investigated using probes such as Zinpyr-1 at time-points later than 1 h after stimulation. Nevertheless, it can still be detected if total zinc is analyzed. Our data are also concurring with the findings of Gaetke et al. They showed that the injection of LPS to healthy human volunteers leads to an instant decrease of serum zinc [27], thus, illustrating that extracellular zinc might be detracted from pathogens in cases of inflammation. This phenomenon was already shown for human hepatocytes, where during acute phase response, an increase of intracellular zinc was detected [28]. However, this information is new for human myeloid cells, indicating that those, as part of the innate immune system, are also able to shift zinc from the extracellular space into the cell as a response to an inflammatory event. Calprotectin levels are highly elevated during various inflammatory diseases. Reports describe pro-inflammatory [29] as well as antimicrobial properties of calprotectin mediated by sequestration of zinc [14]. Elevated expression of calprotectin mRNA has for example been found previously in blood cells during sepsis paralleling serum hypozincemia in a murine model [8]. These findings are in line with our results for human myeloid cell lines. Our real-time PCR data for MM1 cells reveal that both zinc deficiency as well as stimulation with LPS alone are able to significantly enhance the mRNA expression of calprotectin´s subunits S100A8 and S100A9. A rise of calprotectin mRNA expression as a response to an inflammatory condition is corresponding well with previous findings that elevated serum levels of calprotectin can be found in individuals experiencing bacterial infections [12]. Further, we could show that zinc deficiency significantly augmented LPS-induced calprotectin mRNA expression in MM1. Additionally, we could demonstrate this for protein expression, thereby indicating that zinc deficiency and LPS activate different pathways, which synergistically induce high calprotectin expression in CD14 positive MM1 cells. Prior reports of Zwadlo et al. and Odink et al. [30,31] pointed out that S100A8 and S100A9 expression depends on the stage of myeloid cell differentiation. Both proteins are especially expressed in early stages in the myeloid lineage. Strikingly, only S100A9 is expressed under acute inflammatory circumstances in tissue macrophages [30,31]. Thus, this could match our experimental settings of rather acute inflammation. 111
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indicates that CD14 is not necessary to confer zinc’s effects and underlines our hypothesis that zinc deficiency and LPS activate two distinct pathways in myeloid cells. Altogether, the three human cell lines investigated here showed that hypozincemia upregulates calprotectin expression in human myeloid cells. Our results suggest indeed a direct association of zinc homeostasis with calprotectin induction and an independence of the effect of zinc deficiency from pathways activated by LPS via CD14-dependent pathways. As calprotectin binds zinc, a feedback mechanism of zinc levels on calprotectin expression is likely, which should be examined in more detail. Calprotectin levels are highly upregulated during sepsis [36] and seem to play an essential role in the pathophysiology of several inflammatory diseases. It is known that calprotectin is an endogenous ligand of TLR-4 [21], which sustains the secretion of pro-inflammatory cytokines like TNFα [36–38]. This again is hold responsible to be a major factor for the development of sepsis, tissue destruction [39] and the detrimental outcome [40]. Precise adjustment of zinc homeostasis would therefore be a potent mean to prevent or balance a hyper-inflammatory response for instance in systemic inflammatory diseases. However, further studies are needed to examine the exact range of zinc that is needed to balance zinc homeostasis in humans in vivo, especially during disease settings.
cytokines, Am. J. Physiol. Endocrinol. Metab. 285 (5) (2003) E1095–E1102. [11] B.D. Corbin, E.H. Seeley, A. Raab, J. Feldmann, M.R. Miller, V.J. Torres, K.L. Anderson, B.M. Dattilo, P.M. Dunman, R. Gerads, R.M. Caprioli, W. Nacken, W.J. Chazin, E.P. Skaar, Metal chelation and inhibition of bacterial growth in tissue abscesses, Science 319 (5865) (2008) 962–965. [12] I. Striz, I. Trebichavsky, Calprotectin - a pleiotropic molecule in acute and chronic inflammation, Physiol. Res. 53 (3) (2004) 245–253. [13] J. Goyette, C.L. Geczy, Inflammation-associated S100 proteins: new mechanisms that regulate function, Amino Acids 41 (4) (2011) 821–842. [14] C. Kerkhoff, M. Klempt, C. Sorg, Novel insights into structure and function of MRP8 (S100A8) and MRP14 (S100A9), Biochim. Biophys. Acta 1448 (2) (1998) 200–211. [15] D.J. Mazzatti, P. Uciechowski, S. Hebel, G. Engelhardt, A.J. White, J.R. Powell, L. Rink, H. Haase, Effects of long-term zinc supplementation and deprivation on gene expression in human THP-1 mononuclear cells, J. Trace Elem. Med. Biol. 22 (4) (2008) 325–336. [16] L.S. Mayer, P. Uciechowski, S. Meyer, T. Schwerdtle, L. Rink, H. Haase, Differential impact of zinc deficiency on phagocytosis, oxidative burst, and production of proinflammatory cytokines by human monocytes, Metallomics 6 (7) (2014) 1288–1295. [17] S.C. Burdette, G.K. Walkup, B. Spingler, R.Y. Tsien, S.J. Lippard, Fluorescent sensors for Zn(2+) based on a fluorescein platform: synthesis, properties and intracellular distribution, J. Am. Chem. Soc. 123 (32) (2001) 7831–7841. [18] H. Haase, S. Hebel, G. Engelhardt, L. Rink, Flow cytometric measurement of labile zinc in peripheral blood mononuclear cells, Anal. Biochem. 352 (2) (2006) 222–230. [19] K.A. Zarember, P.J. Godowski, Tissue expression of human toll-like receptors and differential regulation of toll-like receptor mRNAs in leukocytes in response to microbes, their products, and cytokines, J. Immunol. 168 (2) (2002) 554–561. [20] M. Maywald, L. Rink, Zinc homeostasis and immunosenescence, J. Trace Elem. Med. Biol. 29 (2015) 24–30. [21] T. Vogl, K. Tenbrock, S. Ludwig, N. Leukert, C. Ehrhardt, M.A. van Zoelen, W. Nacken, D. Foell, T. van der Poll, C. Sorg, J. Roth, Mrp8 and Mrp14 are endogenous activators of toll-like receptor 4, promoting lethal, endotoxin-induced shock, Nat. Med. 13 (9) (2007) 1042–1049. [22] W. Maret, Zinc biochemistry: from a single zinc enzyme to a key element of life, Adv. Nutr. 4 (1) (2013) 82–91. [23] H. Haase, L. Rink, Multiple impacts of zinc on immune function, Metallomics 6 (7) (2014) 1175–1180. [24] H. Haase, J.L. Ober-Blobaum, G. Engelhardt, S. Hebel, A. Heit, H. Heine, L. Rink, Zinc signals are essential for lipopolysaccharide-induced Signal transduction in monocytes, J. Immunol. 181 (9) (2008) 6491–6502. [25] N.Z. Gammoh, L. Rink, Zinc in infection and inflammation, Nutrients 9 (6) (2017). [26] H. Haase, L. Rink, Functional significance of zinc-related signaling pathways in immune cells, Annu. Rev. Nutr. 29 (2009) 133–152. [27] L.M. Gaetke, C.J. McClain, R.T. Talwalkar, S.I. Shedlofsky, Effects of endotoxin on zinc metabolism in human volunteers, Am. J. Physiol. 272 (6 Pt 1) (1997) E952–E956. [28] J.P. Liuzzi, L.A. Lichten, S. Rivera, R.K. Blanchard, T.B. Aydemir, M.D. Knutson, T. Ganz, R.J. Cousins, Interleukin-6 regulates the zinc transporter Zip14 in liver and contributes to the hypozincemia of the acute-phase response, Proc. Natl. Acad. Sci. U. S. A. 102 (19) (2005) 6843–6848. [29] K. Otsuka, F. Terasaki, M. Ikemoto, S. Fujita, B. Tsukada, T. Katashima, Y. Kanzaki, K. Sohmiya, T. Kono, H. Toko, M. Fujita, Y. Kitaura, Suppression of inflammation in rat autoimmune myocarditis by S100A8/A9 through modulation of the proinflammatory cytokine network, Eur. J. Heart Fail. 11 (3) (2009) 229–237. [30] G. Zwadlo, J. Bruggen, G. Gerhards, R. Schlegel, C. Sorg, Two calcium-binding proteins associated with specific stages of myeloid cell differentiation are expressed by subsets of macrophages in inflammatory tissues, Clin. Exp. Immunol. 72 (3) (1988) 510–515. [31] K. Odink, N. Cerletti, J. Bruggen, R.G. Clerc, L. Tarcsay, G. Zwadlo, G. Gerhards, R. Schlegel, C. Sorg, Two calcium-binding proteins in infiltrate macrophages of rheumatoid arthritis, Nature 330 (6143) (1987) 80–82. [32] S. Dubben, A. Honscheid, K. Winkler, L. Rink, H. Haase, Cellular zinc homeostasis is a regulator in monocyte differentiation of HL-60 cells by 1 alpha,25-dihydroxyvitamin D3, J. Leukoc. Biol. 87 (5) (2010) 833–844. [33] K. Triantafilou, M. Triantafilou, R.L. Dedrick, A CD14-independent LPS receptor cluster, Nat. Immunol. 2 (4) (2001) 338–345. [34] H. Haase, L. Rink, Signal transduction in monocytes: the role of zinc ions, Biometals 20 (3–4) (2007) 579–585. [35] S. Gessani, U. Testa, B. Varano, P. Di Marzio, P. Borghi, L. Conti, T. Barberi, E. Tritarelli, R. Martucci, D. Seripa, et al., Enhanced production of LPS-induced cytokines during differentiation of human monocytes to macrophages. Role of LPS receptors, J. Immunol. 151 (7) (1993) 3758–3766. [36] A. Achouiti, T. Vogl, C.F. Urban, M. Rohm, T.J. Hommes, M.A. van Zoelen, S. Florquin, J. Roth, C. van’ t Veer, A.F. de Vos, T. van der Poll, Myeloid-related protein-14 contributes to protective immunity in gram-negative pneumonia derived sepsis, PLoS Pathog. 8 (10) (2012) e1002987. [37] K. Vandal, P. Rouleau, A. Boivin, C. Ryckman, M. Talbot, P.A. Tessier, Blockade of S100A8 and S100A9 suppresses neutrophil migration in response to lipopolysaccharide, J. Immunol. 171 (5) (2003) 2602–2609. [38] H. Huang, L. Tu, Expression of S100 family proteins in neonatal rats with sepsis and its significance, Int. J. Clin. Exp. Pathol. 8 (2) (2015) 1631–1639. [39] B. Sampson, M.K. Fagerhol, C. Sunderkotter, B.E. Golden, P. Richmond, N. Klein, I.Z. Kovar, J.H. Beattie, B. Wolska-Kusnierz, Y. Saito, J. Roth, Hyperzincaemia and hypercalprotectinaemia: a new disorder of zinc metabolism, Lancet 360 (9347) (2002) 1742–1745. [40] H.P. Wu, C.K. Chen, K. Chung, J.C. Tseng, C.C. Hua, Y.C. Liu, D.Y. Chuang, C.H. Yang, Serial cytokine levels in patients with severe sepsis, Inflamm. Res. 58 (7) (2009) 385–393.
5. Conclusion In our experimental setting, zinc deficiency alone enhances the mRNA and protein expression of calprotectin in human myeloid cells independently of maturity stage. Pre-existing zinc deficiency augmented LPS-induced calprotectin expression in CD14+ MM1 but not in CD14− THP-1 or U937. Zinc deficiency and LPS seem therefore to activate different intracellular pathways, offering an explanation for the highly elevated expression levels during severe disease, when pathogen load is high and serum hypozincemia is pronounced. This suggests the trace metal as a new treatment option for diseases with deregulated calprotectin expression and an approach to balance dysregulated inflammatory responses. Conflict of interest None. Acknowledgements L.R. is member of Zinc Net. We thank Gabriela Engelhardt and Silke Hebel for excellent technical assistance. References [1] H. Haase, E. Mocchegiani, L. Rink, Correlation between zinc status and immune function in the elderly, Biogerontology 7 (5–6) (2006) 421–428. [2] L. Rink, H. Haase, Zinc homeostasis and immunity, Trends Immunol. 28 (1) (2007) 1–4. [3] P.J. Fraker, M.E. Gershwin, R.A. Good, A. Prasad, Interrelationships between zinc and immune function, Fed. Proc. 45 (5) (1986) 1474–1479. [4] D. Beyersmann, H. Haase, Functions of zinc in signaling, proliferation and differentiation of mammalian cells, Biometals 14 (3–4) (2001) 331–341. [5] I. Wessels, H. Haase, G. Engelhardt, L. Rink, P. Uciechowski, Zinc deficiency induces production of the proinflammatory cytokines IL-1beta and TNFalpha in promyeloid cells via epigenetic and redox-dependent mechanisms, J. Nutr. Biochem. 24 (1) (2013) 289–297. [6] I. Wessels, J. Jansen, L. Rink, P. Uciechowski, Immunosenescence of polymorphonuclear neutrophils, Sci. WorldJ. 10 (2010) 145–160. [7] H. Haase, L. Rink, The immune system and the impact of zinc during aging, Immun. Ageing 6 (2009) 9. [8] I. Wessels, R.J. Cousins, Zinc dyshomeostasis during polymicrobial sepsis in mice involves zinc transporter Zip14 and can be overcome by zinc supplementation, Am. J. Physiol. Gastrointest. Liver Physiol. 309 (9) (2015) G768–G778. [9] J. Roth, T. Vogl, C. Sorg, C. Sunderkotter, Phagocyte-specific S100 proteins: a novel group of proinflammatory molecules, Trends Immunol. 24 (4) (2003) 155–158. [10] B. Bao, A.S. Prasad, F.W. Beck, M. Godmere, Zinc modulates mRNA levels of
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The role of zinc in calprotectin expression in human myeloid cells Simone Lienau, Lothar Rink, Inga Wessels
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T
Institute of Immunology, Medical Faculty, RWTH Aachen University, Pauwelsstr. 30, D-52074, Aachen, Germany
A R T I C LE I N FO
A B S T R A C T
Keywords: Zinc deficiency Calprotectin Myeloid cells Monocytic cells Inflammation Inflammatory disease
Elevated levels of calprotectin and other inflammatory mediators have been observed in inflammatory diseases paralleling serum hypozincemia. While a role of zinc in the regulation of tumor necrosis factor α, interleukin (IL)-1β and IL-6 expression has been established, the direct interrelation of zinc and calprotectin (S100A8/ S100A9 heterodimer) expression is so far missing. In the present study, we analyzed mRNA and protein levels of S100A8 and S100A9 in monocytic Mono Mac (MM)1 and early myeloid THP-1 and U937 cells to elucidate the effect of zinc deficiency on their expression. We could depict that zinc deficiency alone enhances mRNA and protein expression of calprotectin in myeloid cells, independently from maturity stage. Moreover, pre-existing zinc deficiency augmented lipopolysaccharide (LPS)-induced calprotectin expression in CD14+ MM1, but not in CD14− U937 or CD14− THP-1 cells. Zinc deficiency and LPS seem therefore to activate different intracellular pathways. Our findings suggest that zinc does not only regulate the activity of calprotectin but also its expression by human myeloid cells.
1. Introduction
detail so far, but is important to understand to develop new treatment strategies for these diseases. Hence, the aim of this study was to investigate the influence of zinc homeostasis on calprotectin expression in human myeloid cells of different maturity stages.
A balanced zinc homeostasis is crucial for an appropriate immune response [1]. Zinc deficiency caused by infections, malnutrition, age or diseases may therefore lead to complex immune impairments [2–5]. In the elderly, zinc deficiency was associated with constantly elevated inflammatory parameters [6] and increasing rates of infectious diseases with high mortality therefrom [5,7]. Generally, severe and chronic serum hypozincemia are accompanied by an increase of pro-inflammatory mediators like tumor necrosis factor α, interleukin (IL)-1β, IL-6 and calprotectin [5,8–10]. Calprotectin is a heterodimeric complex belonging to the S100 family, consisting of the subunits S100A8 and S100A9. It can influence zinc metabolism by chelating zinc in the presence of calcium. Amongst other roles, it thereby deprives bacteria of zinc, conveying antimicrobial properties [11]. Predominantly it is expressed by cells of the innate immune system including neutrophil granulocytes and monocytes [12]. Hallmarks of inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease, cystic fibrosis or sepsis are elevated levels of calprotectin in serum paralleled by serum hypozincemia [8,9,13,14]. A prior report links zinc deficiency to the upregulation of calprotectin [15]. The precise association of zinc with calprotectin expression especially in an inflammatory context has not been investigated in
2. Methods and materials 2.1. Cell culture MM1, THP-1 and U937 cells were cultivated at 37 °C, humidified atmosphere and 5% CO2. Culture medium for U937 was RPMI 1640 (Sigma-Aldrich, München, Germany) with 10% heat-inactivated, lowendotoxin FCS (PAA, Cölbe, Germany), 100 U/ml penicillin (SigmaAldrich), 100 μg/ml streptomycin (Sigma-Aldrich) and 2 mM L-glutamine (Sigma-Aldrich). MM1 cells were cultured in U937 medium where 1% non-essentialamino acids (NEAA, Lonza, Köln, Germany) and 1% sodium pyruvate (Lonza) were added. To culture THP-1 cells, 2.5 μl β-Mercaptoethanol (Merck, Darmstadt, Germany) were added per 500 ml of U937 medium. For zinc-deficient medium, zinc adequate control medium with 10% FCS was treated with CHELEX 100 ion exchange resin (Sigma-Aldrich) for 1 h at 20 °C and was subsequently reconstituted with 500 μM CaCl2
Abbreviations: AAS, atomic absorption spectrometry; IL, interleukin; LPS, lipopolysaccharide; MT, metallothionein; MM1, Mono Mac 1; TNF, tumor necrosis factor; TPEN, N,N,N′,N′tetrakis(2-pyridylmethyl)ethylenediamine ⁎ Corresponding author. E-mail address: [email protected] (I. Wessels). https://doi.org/10.1016/j.jtemb.2018.04.022 Received 12 January 2018; Received in revised form 2 April 2018; Accepted 19 April 2018 0946-672X/ © 2018 Elsevier GmbH. All rights reserved.
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(Sigma-Aldrich) and 400 μM MgCl2 (Merck). The metal composition of RPMI 1640 medium with 10% FCS prior and after treatment with CHELEX resin can be found in Mayer et al. [16]. The cells were pre-cultivated for 24 h either in zinc adequate control medium, in zinc deficient medium (CHELEX) as described above, or in zinc reconstituted CHELEX-medium (CHELEX + Zn) with ZnSO4 (ZnSO4 × 7 H2O, Merck) to yield a final concentration of 8 μM. Subsequently lipopolysaccharide (LPS), E. coli serotype O111:B4 (Sigma-Aldrich) was added for another 24 h treatment as indicated in the figure legends.
SYBR Green PCR Master Mix (Applied Biosystems). Fold-changes were calculated through the ΔΔ CT method. 2.7. Cell extracts and Western blotting A total of 1 × 106 cells were lysed in 100 μl lysis buffer (0.5 M Tris–HCl (pH 6.8) (Carl Roth, Karlsruhe, Germany), 2% (w/v) sodium dodecyl sulfate (Merck), 1 mM sodium vanadate (Sigma-Aldrich), 26.6% glycerol (Carl Roth), 1% (v/v) β-mercaptoethanol, 0.01% (w/v) bromophenol blue (Fluka, Buchs, Switzerland)). The cells were sonicated for 15 s and then boiled for 3 min at 95 °C. An equivalent of 2 × 105 cells per lane was separated on 15% polyacrylamide (S100A9 and S100A8) or 10% polyacrylamide gels (βactin) at 150 V and blotted to nitrocellulose membranes. Homogeneous loading of membranes was confirmed with Ponceau S staining (SigmaAldrich). Following destaining, membranes were blocked with tris buffered saline (TBS, 20 mM Tris-HCl (pH 7.6, Carl Roth), 136 mM NaCl (Merck)) including 0.1% (v/v) Tween 20 (AppliChem, Darmstadt, Germany)) and 3% bovine serum albumin (Fluka) and incubated overnight with the primary antibodies against S100A9, S100A8 (Santa Cruz Biotechnology, Heidelberg, Germany) or β-actin (Cell Signaling Technology, Frankfurt am Main, Germany) at 1:200 dilutions in TBS-T containing 5% bovine serum albumin. Membranes were then incubated with horseradish peroxidase (HRP)-linked anti-rabbit IgG secondary antibody and HRP-coupled anti-biotin antibody for detection of biotinlabeled molecular weight (MW) standard (anti-biotin 1:1000, antirabbit 1:2000; Cell Signaling Technology) for 2 h. This was followed by detection with LumiGlo reagent (Cell Signaling Technology) on a LAS3000 (Fujifilm Lifescience, Düsseldorf, Germany).
2.2. Measurement of free intracellular zinc with ZinPyr-1 Free intracellular zinc was measured using ZinPyr-1 (10 μM, Santa Cruz Biotechnology, Heidelberg, Germany). The mean fluorescence signal was detected by FACSCalibur (BD Biosciences, Heidelberg, Germany) using Cellquest software 3.0. The formula [Zn] = KD× [(F − Fmin)/(Fmax − F)] was used to calculate the concentration of free labile intracellular zinc. In doing so, the dissociation constant (KD) was 0.7 nM for the Zn/ZinPyr-1 complex [17]. The maximal (Fmax) and minimal (Fmin) mean fluorescence signals were generated by addition of zinc (100 μM) combined with pyrithione (50 μM) (Sigma-Aldrich) or N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (50 μM) (TPEN, Sigma-Aldrich), respectively [18]. 2.3. ELISA Supernatants were harvested after stimulation, stored at −20 °C until measurement, and only thawed once for cytokine detection. IL-6 protein production was quantified using OptEIA assays (BD Biosciences) according to the manufacturer’s instructions.
2.8. Statistical analysis Statistical significance of the results was analyzed by GraphPad Prism software version 5 (GraphPad software, La Jolla, CA, USA). In the case of Gaussian distribution, as tested by D’Agestino’s K-square test of normality, student’s t-test for unpaired samples was performed. Without this assumption, Mann-Whitney-U test was performed for unpaired samples and Wilcoxon signed rank test was performed for paired samples. p-values ≤ 0.05 were regarded statistically significant and are illustrated in the figures by *, p-values ≤ 0.01 are shown by ** and pvalues ≤ 0.001 are shown by ***.
2.4. Flow cytometry Cells were stained with phycoerythrin (PE)-conjugated CD14 monoclonal antibody (mAbs; BD Biosciences) or with the isotypic control PE-conjugated immunoglobulin IgG2bκ mAb (BD Biosciences) for 15 min. Washed cells were then analyzed in a FACSCalibur (BD Biosciences) using Cellquest software 3.0. 2.5. Atomic absorption spectrometry (AAS)
3. Results Cells were washed with washing buffer (0.9% sodium chloride (NaCl, Merck), 10 mM ethylenediaminetetraacetic acid (EDTA, Merck) and 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES, AppliChem, Darmstadt, Germany)) twice and scraped into icecold water. Subsequently, cells were hydrolyzed in 200 μl HNO3 (> 69.5%, for trace analyses, Sigma-Aldrich) for 2 h at 80 °C. Cellular zinc concentration was measured by flame atomic absorption spectrometry (AAS, Perkin Elmer, Baesweiler, Germany) using a standard curve made from AAS grade zinc standard solution. Absorbance values were normalized to cell counts.
3.1. Zinc homeostasis of MM1 cells during zinc deficiency and after LPSstimulation Initially, we confirmed that pre-incubation in CHELEX-treated medium decreased intracellular free zinc levels in MM1 cells (Fig. 1A), to verify our zinc deficiency model [15,16]. Intracellular free zinc was decreased significantly in MM1 cells by 58.2% to a mean of 0.028 nM (Fig. 1A) incubated in CHELEX-treated medium compared with the zinc adequate controls. However, intracellular zinc levels of zinc adequate controls and zinc deficient MM1 cells were not affected significantly by LPS stimulation for 24 h, compared with the matching unstimulated control. In order to eliminate artefacts from CHELEX resins, we used a second control sample, which comprised of CHELEX-treated medium, replenished with zinc to yield a final concentration of 8 μ M zinc, comparable to regular zinc adequate medium. No significant difference was detected between MM1 cells incubated in regular zinc adequate control medium or in zinc replenished CHELEX-treated medium (Fig. 1B). Due to simplification, only one control group, i.e. cells cultured in regular medium, was used consequentially during further experiments. To support our data for the intracellular free zinc measurements, we used atomic absorption spectrometry to determine total zinc levels in
2.6. Reverse transcription and real-time PCR RNA was isolated using the NucleoSpin® RNA Kit (Macherey-Nagel, Düren, Germany) and cDNA was acquired through use of qScriptTM cDNA Synthesis Kit (Quanta Biosciences, Darmstadt, Germany) according to the manufacturers´ protocols. The primers for S100A8 and S100A9 [15] and the primers for the housekeeping gene glycerinaldehyde 3-phosphate dehydrogenase (GAPDH) [19] were used as previously described. All samples were run using a Step-1 plus (Applied Biosystems, Darmstadt, Germany) with the following parameters: 95 °C for 15 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min for S100A8 and GAPDH and 61 °C for S100A9 in duplicate, using Power 107
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Fig. 1. Zinc homeostasis of myeloid cells during zinc deficiency and after LPS-stimulation. (A) MM1 cells were either pre-incubated in zinc deficient medium (CHE), or in zinc adequate control medium for 24 h with or without subsequent LPS (250 ng/ml) stimulation for 24 h. Intracellular free zinc was detected by flow cytometry using the fluorescent zinc probe ZinPyr-1. Charted are mean values ± SD from n = 5 independent experiments. Statistical significances, quantified by Mann-Whitney U test, are illustrated (*, p ≤ 0.5). (B) The same experiment as in (A) was performed for MM1 comparing the values for the zinc adequate control medium with the values for CHELEXtreated medium, where zinc was replenished to yield a final concentration of 8 μ M. Cells were additionally stimulated with LPS as indicated (250 ng/ml, 24 h). All values are shown as mean ± SD from n = 5 independent experiments. No significant differences have been detected between the two comparable pairs by Mann-Whitney U test. (C, D) Total cellular zinc levels of MM1 cells (C) or their supernatants (D) cultured as in (A) were measured via atomic absorption spectrometry. The bars represent mean values ± SD from (C) n = 9 and (D) n = 4 independent experiments. Statistical significances, calculated by Mann-Whitney U test, are depicted (*, p ≤ 0.5; **, p ≤ 0.01; ***, p ≤ 0.001).
3.3. Zinc deficiency and LPS stimulation cumulatively activate calprotectin protein expression in MM1 cells
MM1 cells. Compared with the control, total cellular zinc levels were significantly decreased by 20.6% to a mean of 0.116 mg/103 cells in cells cultured in CHELEX-treated medium (Fig. 1C). Cells cultured in CHELEX-treated medium may thus further on be called zinc deficient. In contrast to intracellular free zinc levels, total cellular zinc as determined via AAS analysis was significantly increased by LPS stimulation by 21.9% to a mean of 0.178 mg/103 cells (Fig. 1C). In accordance with these findings, zinc levels in the supernatant were significantly decreased in case of LPS stimulation in contrast to the untreated control (Fig. 1D) and by 33.3% to a mean of 0.002 mg/103 cells in case of LPS stimulation after pre-incubation in zinc deficiency in contrast with the corresponding control. Our findings confirmed that incubation in zinc deficient medium decreases free intracellular as well as total cellular zinc in myeloid cells.
Subsequently, we explored calprotectin protein expression in MM1 cells using the same experimental settings. Western blotting (Fig. 2C) and densitometry (Fig. 2D, E) analyses revealed, similar to what was observed for mRNA expression, that zinc deficiency alone significantly increased S100A9 protein levels more than two fold-change (S100A9/ β-actin) compared with the zinc adequate controls (Fig. 2E). Further, LPS stimulation alone significantly activated S100A9 protein expression doubling it as well, which was then significantly further augmented by pre-existing zinc deficiency (Fig. 2E). Trends for S100A8 protein expression were similar (Fig. 2D), but did not reach significance, although elevation of mRNA levels was observed (Fig. 2A). Taken together, our data shows increased calprotectin expression in zinc deprived circumstances in MM1 cells.
3.2. Calprotectin mRNA expression by MM1 cells is up-regulated by zinc deficiency and LPS stimulation
3.4. CD 14 expression and LPS-induced cytokine production is limited to MM1 cells
An association between zinc homeostasis and calprotectin expression has been suggested, but was not investigated in detail so far. Thus, we examined the effects of zinc deficiency on the regulation of calprotectin mRNA expression, with or without the presence of LPS stimulation to model an infection, by real-time PCR. After cultivation in zinc deficient medium, the expression of S100A8 and S100A9 mRNA in MM1 cells was significantly elevated reaching over 5 fold (S100A8 mRNA/GAPDH) and 3 fold (S100A9 mRNA/GAPDH) the expression of zinc adequate controls (Fig. 2A, B). Furthermore, stimulation of zinc adequate controls with LPS for 24 h significantly activated S100A8 and S100A9 mRNA expression by 998% to a mean fold-change of 10.98 (S100A8 mRNA/GAPDH) and by 679.6% to a mean fold-change of 7.796 (S100A9 mRNA/GAPDH). Zinc deficiency preceding LPS stimulation significantly boosted LPS-induced mRNA expression of S100A8 and S100A9 in MM1 cells, compared to zinc deficiency or LPS stimulation alone. These data revealed that zinc deficiency leads to a rise of calprotectin mRNA expression in monocytic MM1 cells and augments its expression during inflammatory settings.
Results from analyses in MM1 cells showed a synergistic effect of zinc deficiency and LPS stimulation on the expression of calprotectin. This suggests that independent pathways may be activated here by zinc deficiency compared to LPS stimulation. In order to distinguish effects mediated via CD14-dependent pathways from processes mediated via alternative pathways, we examined THP-1 and U937 cells, which do not express significant amounts of CD14 on their surface as verified by flowcytometric investigation and compared results to those from MM1 cells, positive for CD14 expression (Fig. 3A–C). In addition, only MM1 cells were able to secrete significant amounts of IL-6 after LPS stimulation, whereas no significant production was detected for THP-1 and U937 cells (Fig. 3D) confirming that CD14-dependent signaling pathways are not activated by LPS in U937 and THP-1. This rendered THP-1 and U937 ideal for investigating CD14-independent pathways. Investigation of alternative cell lines also enabled us to generate a more representative model for human myeloid cells, to eliminate cell line specific effects, and effects elicited by the 108
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Fig. 2. Zinc-homeostasis is involved in the regulation of calprotectin mRNA and protein expression in myeloid cell lines. MM1 cells were pre-incubated in zinc adequate control medium or zinc deficient medium (CHE) for 24 h and subsequently stimulated with LPS (250 ng/ml, 24 h). (A, B) mRNA from MM1 cells was collected, reverse transcribed and quantified by real-time PCR using SBYR-Green. The gene expression of S100A8 (A) and S100A9 (B) was normalized to the housekeeping gene GAPDH. The fold changes were calculated by the ΔΔ CT-method for n = 7 independent experiments. Shown are mean values ± SD. Statistically significant differences are marked (*, p ≤ 0.5; **, p ≤ 0.01) as quantified by Mann-Whitney-U test. (C, D, E) Human calprotectin protein expression in MM1 cells was analyzed by western blotting with antibodies against S100A8, S100A9 and β-actin (C). The relative density of the protein bands of S100A8 (D) and of S100A9 (E) was quantified with regard to the control β-actin. For (D) n = 4 and for (E) n = 13 independent experiments were performed and values are shown as mean ± SD. Statistically significant differences are marked (*, p ≤ 0.5; **, p ≤ 0.01; ***, p ≤ 0.001) as quantified by Mann-Whitney-U test.
degeneration of a certain tumor cell line.
3.6. Regulation of calprotectin mRNA expression in THP-1 and U937 cells by zinc deficiency
3.5. Zinc homeostasis of THP-1 and U937 cells during zinc deficiency and after LPS-stimulation resembles findings for MM1 cells
Subsequently, we examined the expression of S100A8 and S100A9 mRNA in THP-1 and U937 cells. Contrasting the findings in MM1 cells (Fig. 2A, B), LPS stimulation alone was not able to significantly induce S100A8 and S100A9 mRNA expression in both THP-1 (Fig. 5A, B) and in U937 (Fig. 5C, D) cells compared with the unstimulated control. However, a rise in the mRNA expression of S100A8 and S100A9 after cultivation in a zinc deficient environment was observed in comparison with the control (Fig. 5A–D). This was significantly increased for S100A8 mRNA of THP-1 cells. Hence, our results demonstrate that zinc deficiency increases the expression of calprotectin in all examined human myeloid cell lines.
Experiments with the rather immature myeloid cell lines THP-1 and U937 were done in a uniform manner as for the MM1 cells. Fig. 4 illustrates our findings that the intracellular zinc content in THP-1 (Fig. 4A) and in U937 (Fig. 4B) was significantly decreased by 57.3% to a mean zinc concentration of 0.053 nM and 41.9% to a mean of 0.036 nM respectively in cells cultured in zinc deficient medium for 24 h in comparison with the respective control. No significant differences in the intracellular zinc content could be demonstrated for these cell lines after LPS stimulation for further 24 h in comparison with the matching control. This is in accordance with our findings for MM1 cells.
4. Discussion This present study provides evidence for a regulatory role of zinc homeostasis not only in calprotectin activity, but also in its expression 109
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Fig. 3. Analysis of CD 14 expression on the surface of human cell lines as a marker for myeloid differentiation. Flow cytometry was used to detect the expression of CD14 on the surface of MM1 (A), THP-1 (B) and U937 cells (C). Shown is one representative example of three individual experiments, respectively. The graphs show the counts of CD14 positive cells in comparison to the matching isotypic control phycoerythrin-conjugated immunoglobulin IgG2bκ mAb. Furthermore, zinc adequate U937, THP-1 and MM1 cells were stimulated with LPS (100 ng/ml, 24 h) and IL-6 secretion was analyzed in their supernatants. (D) presents n = 6 independent ELISA’s and values are shown as mean ± SD. Statistical significances, calculated by Mann-Whitney U test, are depicted (*, p ≤ 0.5).
LPS stimulation for 24 h did not show a long-term effect on the measured intracellular zinc levels in these cells. Previous studies by Haase et al. revealed an instant increase of free intracellular zinc within minutes after treatment with LPS. They could show that nearly normal zinc levels were recovered in less than one hour post stimulation [24]. Our time of measurement was 24 h after the stimulation with LPS. This suggests that the short-term effect of a LPS-induced increase of free zinc, a so-called zinc flux, has already been normalized at the time of our measurement, explaining why no effects of LPS on intracellular free zinc levels were observed here. AAS measurements of total cellular zinc confirmed the decrease of the intracellular free zinc levels in MM1 cells after incubation in zinc deficient medium. This indicates that in response to zinc deficient conditions, myeloid cells do loose the trace element and do not only redistribute it into cell organelles or attach it to zinc binding proteins like metallothionein in case of zinc deficiency. In contrast, stimulation with LPS led to a significant rise in total cellular zinc and to a corresponding significant drop of total zinc in the supernatant. One can hypothesize that zinc is taken up by the cell, shortly after LPS stimulation, as suggested by Haase et al. [26]. Subsequently, zinc is probably stored in compartments or tightly bound to zinc-binding proteins, rendering it undetectable if free zinc is
in human myeloid cell lines. More precisely, zinc deficiency alone was shown to induce calprotectin expression in our experimental setting and to augment LPS-induced calprotectin expression. Zinc deficiency may be classified depending on its severity. Slight zinc deficiency can be regularly found in the elderly causing mild immune disturbances [20]. Severe zinc deficiency was correlated with overshooting immune responses and elevated levels of calprotectin in sepsis patients and associated with sepsis initiation and progression in these patients [21]. Although intracellular free zinc levels only represent a small proportion of the total cellular zinc content [22], free zinc is essential for a plurality of cellular processes like intracellular communication [16], second messenger function [23] or regulation of cytokine production [24]. The free intracellular zinc pool is quickly exchangeable, due to the possibility to release zinc promptly from metal binding proteins like metallothionein (MT) [25]. Like MT, Calprotectin is also able to sequester zinc and is expressed mainly by granulocytes and monocytes [12]. Intracellular free zinc concentrations were measured around 0.07 nM in MM1 cells, consistent with former findings [24]. While we could reproduce a decrease of intracellular zinc in the investigated cell lines after pre-incubation in zinc deficient medium [15,16], subsequent
Fig. 4. Zinc homeostasis of THP-1 and U937 during zinc deficiency and after LPS-stimulation. THP-1 (A) and U937 (B) cells were either pre-incubated in zinc deficient (CHE), zinc reconstituted CHELEX-medium (CHE + Zn) or zinc adequate control medium for 24 h and stimulated with LPS (250 ng/ml) for additional 24 h. Intracellular free zinc was detected by flow cytometry using the fluorescent zinc probe ZinPyr-1. Charted are mean values ± SD from (A) n = 5 and (B) n = 3–6 independent experiments. Statistical significances, quantified by Mann-Whitney U test, are illustrated (*, p ≤ 0.5; **, p ≤ 0.01). 110
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Fig. 5. Zinc deficiency upregulates calprotectin mRNA expression in THP-1 and U937 cells. Same experiments as in (2A and B) were performed for THP-1 (5A, B) and for U937 (5C, D) cells. In (A) the expression of S100A8 mRNA in THP-1 cells normalized to the housekeeping gene GAPDH is shown, whereas in (5B) the expression of S100A9 mRNA in THP-1 cells is presented. Consistent, the same experiments were implemented for U937. Depicting in (C) is the mRNA expression of S100A8 and in (D) the expression of S100A9 mRNA. Data are shown as mean from n = 9 (A, B) and n = 3 (C, D) independent experiments ± SD. Statistically significant differences, quantified by MannWhitney-test, are marked (*, p ≤ 0.5; **, p ≤ 0.01).
Moreover, our data are in line with the previous findings of Mazzatti et al. They showed that calprotectin subunits S100A8 and S100A9 were upregulated in myeloid THP-1 cells after cultivation with the zinc chelator TPEN (N,N,N',N'-tetrakis(2-pyridyl-methyl)ethylenediamine) for 40 h [15]. We extended the experimental settings by investigating three different myeloid cell lines of different maturity stages and using CHELEX resin instead of TPEN to generate models for pre-existing zinc deficiency. One disadvantage of TPEN is that it was shown to effect cell metabolism directly in addition to effects via its zinc chelating capacity. Additionally, one major benefit of CHELEX resins is that they withdraw zinc from medium with over 90% efficiency and that resins are separated from the medium before the latter is used for cell culture [16]. While Mazzatti et al. [15] investigated only the effect of zinc deficiency, we additionally examined the effect of zinc deficiency in an inflammatory setting and stimulated the cells with LPS for 24 h. Dubben et al. found out that during 1α,25-dihydroxyitamin D3-induced differentiation, the intracellular free zinc levels in the monocytic cell line HL-60 declined while the expression of S100A8 and S100A9 increased [32]. This underlines our finding that calprotectin expression increases with decreasing intracellular zinc levels. Our results from calprotectin mRNA expression analyses in MM1 suggested that independent pathways may be activated by zinc deficiency compared to LPS stimulation. LPS-mediated signaling is well defined and requires mainly activation via CD14 [33]. The surface marker CD14 is expressed on monocytes and further activates the tolllike receptor (TLR)-4 in those cells after binding to LPS. Subsequently it can activate a signal cascade via the nuclear factor kappa-light-chainenhancer of activated B cells (NF-κB), which among others is liable for the secretion of pro-inflammatory cytokines like IL-1β, IL-6 and TNF-α [24,25,34]. However, it has been shown that expression of some proinflammatory mediators cannot be completely blocked by CD14-specific monoclonal antibodies [35], suggesting alternative pathways. In order to discern effects mediated via CD14-induced signaling pathways from other cellular mechanisms, we examined CD14+ MM1 compared to CD14− and LPS-unresponsive THP-1 and U937 cells. CD14 appears to be necessary to enable LPS-induced calprotectin expression. However, CD14− U937 and THP-1 cells were still able to express increased amounts of calprotectin mRNA during zinc deficiency. This
investigated using probes such as Zinpyr-1 at time-points later than 1 h after stimulation. Nevertheless, it can still be detected if total zinc is analyzed. Our data are also concurring with the findings of Gaetke et al. They showed that the injection of LPS to healthy human volunteers leads to an instant decrease of serum zinc [27], thus, illustrating that extracellular zinc might be detracted from pathogens in cases of inflammation. This phenomenon was already shown for human hepatocytes, where during acute phase response, an increase of intracellular zinc was detected [28]. However, this information is new for human myeloid cells, indicating that those, as part of the innate immune system, are also able to shift zinc from the extracellular space into the cell as a response to an inflammatory event. Calprotectin levels are highly elevated during various inflammatory diseases. Reports describe pro-inflammatory [29] as well as antimicrobial properties of calprotectin mediated by sequestration of zinc [14]. Elevated expression of calprotectin mRNA has for example been found previously in blood cells during sepsis paralleling serum hypozincemia in a murine model [8]. These findings are in line with our results for human myeloid cell lines. Our real-time PCR data for MM1 cells reveal that both zinc deficiency as well as stimulation with LPS alone are able to significantly enhance the mRNA expression of calprotectin´s subunits S100A8 and S100A9. A rise of calprotectin mRNA expression as a response to an inflammatory condition is corresponding well with previous findings that elevated serum levels of calprotectin can be found in individuals experiencing bacterial infections [12]. Further, we could show that zinc deficiency significantly augmented LPS-induced calprotectin mRNA expression in MM1. Additionally, we could demonstrate this for protein expression, thereby indicating that zinc deficiency and LPS activate different pathways, which synergistically induce high calprotectin expression in CD14 positive MM1 cells. Prior reports of Zwadlo et al. and Odink et al. [30,31] pointed out that S100A8 and S100A9 expression depends on the stage of myeloid cell differentiation. Both proteins are especially expressed in early stages in the myeloid lineage. Strikingly, only S100A9 is expressed under acute inflammatory circumstances in tissue macrophages [30,31]. Thus, this could match our experimental settings of rather acute inflammation. 111
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indicates that CD14 is not necessary to confer zinc’s effects and underlines our hypothesis that zinc deficiency and LPS activate two distinct pathways in myeloid cells. Altogether, the three human cell lines investigated here showed that hypozincemia upregulates calprotectin expression in human myeloid cells. Our results suggest indeed a direct association of zinc homeostasis with calprotectin induction and an independence of the effect of zinc deficiency from pathways activated by LPS via CD14-dependent pathways. As calprotectin binds zinc, a feedback mechanism of zinc levels on calprotectin expression is likely, which should be examined in more detail. Calprotectin levels are highly upregulated during sepsis [36] and seem to play an essential role in the pathophysiology of several inflammatory diseases. It is known that calprotectin is an endogenous ligand of TLR-4 [21], which sustains the secretion of pro-inflammatory cytokines like TNFα [36–38]. This again is hold responsible to be a major factor for the development of sepsis, tissue destruction [39] and the detrimental outcome [40]. Precise adjustment of zinc homeostasis would therefore be a potent mean to prevent or balance a hyper-inflammatory response for instance in systemic inflammatory diseases. However, further studies are needed to examine the exact range of zinc that is needed to balance zinc homeostasis in humans in vivo, especially during disease settings.
cytokines, Am. J. Physiol. Endocrinol. Metab. 285 (5) (2003) E1095–E1102. [11] B.D. Corbin, E.H. Seeley, A. Raab, J. Feldmann, M.R. Miller, V.J. Torres, K.L. Anderson, B.M. Dattilo, P.M. Dunman, R. Gerads, R.M. Caprioli, W. Nacken, W.J. Chazin, E.P. Skaar, Metal chelation and inhibition of bacterial growth in tissue abscesses, Science 319 (5865) (2008) 962–965. [12] I. Striz, I. Trebichavsky, Calprotectin - a pleiotropic molecule in acute and chronic inflammation, Physiol. Res. 53 (3) (2004) 245–253. [13] J. Goyette, C.L. Geczy, Inflammation-associated S100 proteins: new mechanisms that regulate function, Amino Acids 41 (4) (2011) 821–842. [14] C. Kerkhoff, M. Klempt, C. Sorg, Novel insights into structure and function of MRP8 (S100A8) and MRP14 (S100A9), Biochim. Biophys. Acta 1448 (2) (1998) 200–211. [15] D.J. Mazzatti, P. Uciechowski, S. Hebel, G. Engelhardt, A.J. White, J.R. Powell, L. Rink, H. Haase, Effects of long-term zinc supplementation and deprivation on gene expression in human THP-1 mononuclear cells, J. Trace Elem. Med. Biol. 22 (4) (2008) 325–336. [16] L.S. Mayer, P. Uciechowski, S. Meyer, T. Schwerdtle, L. Rink, H. Haase, Differential impact of zinc deficiency on phagocytosis, oxidative burst, and production of proinflammatory cytokines by human monocytes, Metallomics 6 (7) (2014) 1288–1295. [17] S.C. Burdette, G.K. Walkup, B. Spingler, R.Y. Tsien, S.J. Lippard, Fluorescent sensors for Zn(2+) based on a fluorescein platform: synthesis, properties and intracellular distribution, J. Am. Chem. Soc. 123 (32) (2001) 7831–7841. [18] H. Haase, S. Hebel, G. Engelhardt, L. Rink, Flow cytometric measurement of labile zinc in peripheral blood mononuclear cells, Anal. Biochem. 352 (2) (2006) 222–230. [19] K.A. Zarember, P.J. Godowski, Tissue expression of human toll-like receptors and differential regulation of toll-like receptor mRNAs in leukocytes in response to microbes, their products, and cytokines, J. Immunol. 168 (2) (2002) 554–561. [20] M. Maywald, L. Rink, Zinc homeostasis and immunosenescence, J. Trace Elem. Med. Biol. 29 (2015) 24–30. [21] T. Vogl, K. Tenbrock, S. Ludwig, N. Leukert, C. Ehrhardt, M.A. van Zoelen, W. Nacken, D. Foell, T. van der Poll, C. Sorg, J. Roth, Mrp8 and Mrp14 are endogenous activators of toll-like receptor 4, promoting lethal, endotoxin-induced shock, Nat. Med. 13 (9) (2007) 1042–1049. [22] W. Maret, Zinc biochemistry: from a single zinc enzyme to a key element of life, Adv. Nutr. 4 (1) (2013) 82–91. [23] H. Haase, L. Rink, Multiple impacts of zinc on immune function, Metallomics 6 (7) (2014) 1175–1180. [24] H. Haase, J.L. Ober-Blobaum, G. Engelhardt, S. Hebel, A. Heit, H. Heine, L. Rink, Zinc signals are essential for lipopolysaccharide-induced Signal transduction in monocytes, J. Immunol. 181 (9) (2008) 6491–6502. [25] N.Z. Gammoh, L. Rink, Zinc in infection and inflammation, Nutrients 9 (6) (2017). [26] H. Haase, L. Rink, Functional significance of zinc-related signaling pathways in immune cells, Annu. Rev. Nutr. 29 (2009) 133–152. [27] L.M. Gaetke, C.J. McClain, R.T. Talwalkar, S.I. Shedlofsky, Effects of endotoxin on zinc metabolism in human volunteers, Am. J. Physiol. 272 (6 Pt 1) (1997) E952–E956. [28] J.P. Liuzzi, L.A. Lichten, S. Rivera, R.K. Blanchard, T.B. Aydemir, M.D. Knutson, T. Ganz, R.J. Cousins, Interleukin-6 regulates the zinc transporter Zip14 in liver and contributes to the hypozincemia of the acute-phase response, Proc. Natl. Acad. Sci. U. S. A. 102 (19) (2005) 6843–6848. [29] K. Otsuka, F. Terasaki, M. Ikemoto, S. Fujita, B. Tsukada, T. Katashima, Y. Kanzaki, K. Sohmiya, T. Kono, H. Toko, M. Fujita, Y. Kitaura, Suppression of inflammation in rat autoimmune myocarditis by S100A8/A9 through modulation of the proinflammatory cytokine network, Eur. J. Heart Fail. 11 (3) (2009) 229–237. [30] G. Zwadlo, J. Bruggen, G. Gerhards, R. Schlegel, C. Sorg, Two calcium-binding proteins associated with specific stages of myeloid cell differentiation are expressed by subsets of macrophages in inflammatory tissues, Clin. Exp. Immunol. 72 (3) (1988) 510–515. [31] K. Odink, N. Cerletti, J. Bruggen, R.G. Clerc, L. Tarcsay, G. Zwadlo, G. Gerhards, R. Schlegel, C. Sorg, Two calcium-binding proteins in infiltrate macrophages of rheumatoid arthritis, Nature 330 (6143) (1987) 80–82. [32] S. Dubben, A. Honscheid, K. Winkler, L. Rink, H. Haase, Cellular zinc homeostasis is a regulator in monocyte differentiation of HL-60 cells by 1 alpha,25-dihydroxyvitamin D3, J. Leukoc. Biol. 87 (5) (2010) 833–844. [33] K. Triantafilou, M. Triantafilou, R.L. Dedrick, A CD14-independent LPS receptor cluster, Nat. Immunol. 2 (4) (2001) 338–345. [34] H. Haase, L. Rink, Signal transduction in monocytes: the role of zinc ions, Biometals 20 (3–4) (2007) 579–585. [35] S. Gessani, U. Testa, B. Varano, P. Di Marzio, P. Borghi, L. Conti, T. Barberi, E. Tritarelli, R. Martucci, D. Seripa, et al., Enhanced production of LPS-induced cytokines during differentiation of human monocytes to macrophages. Role of LPS receptors, J. Immunol. 151 (7) (1993) 3758–3766. [36] A. Achouiti, T. Vogl, C.F. Urban, M. Rohm, T.J. Hommes, M.A. van Zoelen, S. Florquin, J. Roth, C. van’ t Veer, A.F. de Vos, T. van der Poll, Myeloid-related protein-14 contributes to protective immunity in gram-negative pneumonia derived sepsis, PLoS Pathog. 8 (10) (2012) e1002987. [37] K. Vandal, P. Rouleau, A. Boivin, C. Ryckman, M. Talbot, P.A. Tessier, Blockade of S100A8 and S100A9 suppresses neutrophil migration in response to lipopolysaccharide, J. Immunol. 171 (5) (2003) 2602–2609. [38] H. Huang, L. Tu, Expression of S100 family proteins in neonatal rats with sepsis and its significance, Int. J. Clin. Exp. Pathol. 8 (2) (2015) 1631–1639. [39] B. Sampson, M.K. Fagerhol, C. Sunderkotter, B.E. Golden, P. Richmond, N. Klein, I.Z. Kovar, J.H. Beattie, B. Wolska-Kusnierz, Y. Saito, J. Roth, Hyperzincaemia and hypercalprotectinaemia: a new disorder of zinc metabolism, Lancet 360 (9347) (2002) 1742–1745. [40] H.P. Wu, C.K. Chen, K. Chung, J.C. Tseng, C.C. Hua, Y.C. Liu, D.Y. Chuang, C.H. Yang, Serial cytokine levels in patients with severe sepsis, Inflamm. Res. 58 (7) (2009) 385–393.
5. Conclusion In our experimental setting, zinc deficiency alone enhances the mRNA and protein expression of calprotectin in human myeloid cells independently of maturity stage. Pre-existing zinc deficiency augmented LPS-induced calprotectin expression in CD14+ MM1 but not in CD14− THP-1 or U937. Zinc deficiency and LPS seem therefore to activate different intracellular pathways, offering an explanation for the highly elevated expression levels during severe disease, when pathogen load is high and serum hypozincemia is pronounced. This suggests the trace metal as a new treatment option for diseases with deregulated calprotectin expression and an approach to balance dysregulated inflammatory responses. Conflict of interest None. Acknowledgements L.R. is member of Zinc Net. We thank Gabriela Engelhardt and Silke Hebel for excellent technical assistance. References [1] H. Haase, E. Mocchegiani, L. Rink, Correlation between zinc status and immune function in the elderly, Biogerontology 7 (5–6) (2006) 421–428. [2] L. Rink, H. Haase, Zinc homeostasis and immunity, Trends Immunol. 28 (1) (2007) 1–4. [3] P.J. Fraker, M.E. Gershwin, R.A. Good, A. Prasad, Interrelationships between zinc and immune function, Fed. Proc. 45 (5) (1986) 1474–1479. [4] D. Beyersmann, H. Haase, Functions of zinc in signaling, proliferation and differentiation of mammalian cells, Biometals 14 (3–4) (2001) 331–341. [5] I. Wessels, H. Haase, G. Engelhardt, L. Rink, P. Uciechowski, Zinc deficiency induces production of the proinflammatory cytokines IL-1beta and TNFalpha in promyeloid cells via epigenetic and redox-dependent mechanisms, J. Nutr. Biochem. 24 (1) (2013) 289–297. [6] I. Wessels, J. Jansen, L. Rink, P. Uciechowski, Immunosenescence of polymorphonuclear neutrophils, Sci. WorldJ. 10 (2010) 145–160. [7] H. Haase, L. Rink, The immune system and the impact of zinc during aging, Immun. Ageing 6 (2009) 9. [8] I. Wessels, R.J. Cousins, Zinc dyshomeostasis during polymicrobial sepsis in mice involves zinc transporter Zip14 and can be overcome by zinc supplementation, Am. J. Physiol. Gastrointest. Liver Physiol. 309 (9) (2015) G768–G778. [9] J. Roth, T. Vogl, C. Sorg, C. Sunderkotter, Phagocyte-specific S100 proteins: a novel group of proinflammatory molecules, Trends Immunol. 24 (4) (2003) 155–158. [10] B. Bao, A.S. Prasad, F.W. Beck, M. Godmere, Zinc modulates mRNA levels of
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