Copper Regulates Maturation and Expression of an MITF:Tryptase Axis in Mast Cells

Jun Mei Hu Frisk, Lena Kjellén, Stephen G. Kaler, Gunnar Pejler and Helena Öhrvik
J Immunol December 15, 2017, 199 (12) 4132-4141; DOI: https://doi.org/10.4049/jimmunol.1700786

Abstract

Copper has previously been implicated in the regulation of immune responses, but the impact of this metal on mast cells is poorly understood. In this article, we address this issue and show that copper starvation of mast cells causes increased granule maturation, as indicated by higher proteoglycan content, stronger metachromatic staining, and altered ultrastructure in comparison with nontreated cells, whereas copper overload has the opposite effects. In contrast, copper status did not impact storage of histamine in mast cells, nor did alterations in copper levels affect the ability of mast cells to degranulate in response to IgER cross-linking. A striking finding was decreased tryptase content in mast cells with copper overload, whereas copper starvation increased tryptase content. These effects were associated with corresponding shifts in tryptase mRNA levels, suggesting that copper affects tryptase gene regulation. Mechanistically, we found that alterations in copper status affected the expression of microphthalmia-associated transcription factor, a transcription factor critical for driving tryptase expression. We also found evidence supporting the concept that the effects on microphthalmia-associated transcription factor are dependent on copper-mediated modulation of MAPK signaling. Finally, we show that, in MEDNIK syndrome, a condition associated with low copper levels and a hyperallergenic skin phenotype, including pruritis and dermatitis, the number of tryptase-positive mast cells is increased. Taken together, our findings reveal a hitherto unrecognized role for copper in the regulation of mast cell gene expression and maturation.

Introduction

Mast cells are myeloid cells of the hematopoietic system (1, 2). They are characterized by an abundance of highly electron-dense secretory granules that are filled with large quantities of numerous preformed compounds (3). These include histamine and other biogenic amines, serglycin proteoglycans of heparin or chondroitin sulfate type, lysosomal enzymes, and various mast cell–restricted proteases, including tryptase, chymase, and carboxypeptidase A3 (CPA3) (3, 4). When mast cells are activated (e.g., through IgER cross-linking or by engagement of the Mrgprb2/MRGPRX2 receptor), they undergo degranulation, a process associated with the release of these preformed mediators from the granules into the extracellular space (5, 6). Mast cells have been implicated in a variety of pathological settings, most notably including allergic disorders, but there is also a wealth of evidence suggesting that mast cells participate actively in a variety of other types of disease, such as arthritis, cancer, and bacterial infection (7–9).

Copper is essential for normal growth and development, being crucial for many biological processes, including cellular respiration, iron metabolism, and superoxide disproportionation (10–12). Moreover, many human pathologies, such as Alzheimer’s disease, neutropenia, cardiomyopathy, and cancer, are closely associated with defects in copper metabolism (13, 14–18). There are also several monogenic disorders caused by defects in copper metabolism, including MEDNIK syndrome and Menkes disease. Patients with these conditions exhibit severe complications, often with lethal outcomes (19, 20).

Among the biological effects of copper, there is now increased awareness that copper may have a large impact on the immune system. For example, copper is implicated in host defense against pathogens (e.g., bacteria and yeast), and there is evidence supporting that copper can regulate neutrophil and macrophage function (16, 21). With regard to mast cells, it was shown that those lacking Ctr2, a protein implicated in copper transport, exhibited altered homeostasis (22). However, the direct impact of copper on mast cells has not been investigated. We address this issue in this article and show that manipulation of copper levels imposes major effects on mast cell homeostasis, including effects on granular content and on gene expression. Hence, copper emerges as a new factor of importance for regulating mast cell function.

Materials and Methods

Reagents

Copper (II) sulfate and bathocuproinedisulfonic acid disodium salt (BCS) were purchased from Sigma-Aldrich (Steinheim, Germany). Copper and BCS solutions were prepared in dH2O with stock concentrations of 10–20 and 200 mM, respectively. The solutions were filtered through 0.2-μM filters before usage. PD98059 was obtained from Sigma-Aldrich. A stock solution of PD98059 was prepared in DMSO.

Animals and cells

C57BL/6 mice (12–15 weeks) were used for experiments. The animal experiments were approved by the local ethical committee (no. C 31/14; Uppsala djurförsöksetiska nämnd, Uppsala, Sweden). Bone marrow–derived mast cells (BMMCs) were established as described (23). The cell cultures were maintained at one half million cells per milliliter, and the medium was changed twice a week. Mature and pure BMMCs were obtained after a 4-wk culture in IL-3–supplemented media. The melanoma cell line B16F10 was cultured in DMEM supplemented with 10% FBS serum.

Skin biopsies

Skin biopsies were obtained from a 2-y-old subject with MEDNIK syndrome under National Institutes of Health Protocol 09-CH-0059, with approval by The Eunice Kennedy Shriver National Institute of Child Health and Human Development Institutional Review Board and informed parental consent. Separately, skin biopsy specimens were obtained from two healthy adult volunteers. Biopsies were fixed in 4% paraformaldehyde/PBS, embedded in paraffin, and stained for chymase using chloroacetate esterase, as previously described (23), tryptase using a mAb (MAB1222; Merck Millipore, Solna, Sweden), and toluidine blue solution (0.1% toluidine blue in 0.17 mM NaCl [pH 2]) for detecting mast cells. Photographs were taken using a Nikon brightfield microscope.

Cell viability test

Cell viability was monitored using the CellTiter-Blue Cell Viability Assay (Promega-Invitrogen). Briefly, 10 μl of cell viability reagent was mixed with 90 μl of cell suspension and incubated for 1 h at 37°C (5% CO2). A fluorescence scan was performed using a microplate reader (TECAN Infinite M200) at 560 nm for excitation and 590 nm for emission. Triplicates or quadruplicates were performed for each measurement. The data were derived from three independent experiments and are presented as the mean ± SD.

Morphological assessments

Cytospin slides were prepared from 50,000 to 75,000 BMMCs per slide. May-Grünwald/Giemsa (Merck) (23) staining and transmission electron microscopy (TEM) analysis were performed as described previously (24) with 5 × 106 BMMCs cultured under normal copper conditions, excess copper (20 μM), or deprived of copper (BCS: 200 μM).

Western blot analysis

Harvested cells were washed twice with ice-cold PBS before protein analysis. The cell pellets were lysed with a 10× volume of lysis buffer 1 (1% Triton X-100, 0.1% SDS, and 1 mM EDTA in PBS [pH 7.4]) or lysis buffer 2 (50 mM Tris, 150 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS, and 1 mM EDTA) in the presence of Pierce phosphatase inhibitor (Thermo Fisher Scientific, Waltham, MA) and protease inhibitor mixture (Roche Diagnostics) for 30–45 min on ice. The cell debris was removed by centrifugation at 14,000 rpm for 20 min at 4°C, and the supernatant was collected for protein analysis. The protein concentration was assessed using a Bicinchoninic Acid Kit (Sigma-Aldrich). Equal amounts of protein were used for Western blot analysis, as described previously (23), and fluorescence-labeled anti-rabbit and anti-goat IgG were used for detection of fluorescence using an Odyssey Infrared Imager (Li-Cor). The following Abs were used: anti-microphthalmia-associated transcription factor (Mitf) (ab12039; Abcam), anti-ERK1/2 and anti–p-ERK1/2 (Cell Signaling Technology, anti-actin (I-19; Santa Cruz Biotechnology), and anti-Ctr1 and anti-Mcpt6 antisera (raised in rabbits) (23, 25). Densitometric scanning was performed using ImageJ software.

Quantitative real-time PCR

Total RNA from cell pellets was isolated according to the instructions provided by the manufacturer (MACHEREY-NAGEL, Düren, Germany). RNA purity and concentration were assessed with a NanoDrop device. One-hundred nanograms of highly purified RNA (A260/280 ≥ 1.95) was used for cDNA synthesis. RNA was reverse transcribed using a purchased kit (Bio-Rad, Solna, Sweden). Five milliliters of SYBR GreenER SuperMix (Invitrogen, Carlsbad, CA), 0.2 μl of Rox reference dye (Invitrogen), 0.2 μl of primer mix, 3.6 μl of dH2O, and 1 μl of cDNA were mixed before running quantitative real-time PCR (qPCR) using a 7900HT Fast Real-Time PCR System (Thermo Fisher Scientific). The program for qPCR was 50°C for 2 min, 95°C for 10 min, 95°C for 15 s, and 60°C for 1 min in 40 cycles and 95°C for 15 s, 60°C for 15 s, and 95°C for 15 s. GAPDH was used as a housekeeping gene. The relative amount of cDNA was determined in duplicates or triplicates and calculated according to the 2−ΔΔCT method. The following primers were used: GAPDH forward: 5′-CTC CCA CTC TTC CAC CTT CG-3′, GAPDH reverse: 5′-CCA CCA CCC TGT TGC TGT AG-3′; Chst11 forward: 5′-CCA AAG TAT GTT GCA CCC AGT-3′, Chst11 reverse: 5′-CTG GTC CCG TCT CAT CTG GT-3′; Ndst2 forward: 5′-GTG GCT GAT GTT GAG GCT TTG-3′, Ndst2 reverse: 5′-ATC CTC CTC TTC TGT CCC GG-3′; Cpa3 forward: 5′-TGA CAG GGA GAA GGT ATT CCG-3′, Cpa3 reverse: 5′-CCA AGG TTG ACT GGA TGG TCT-3′; Mcpt4 forward: 5′-GCA GTC TTC ACC CGA ATC TC-3′, Mcpt4 reverse: 5′-CAG GAT GGA CAC ATG CTT TG-3′; Mcpt5 forward: 5′-CCT GTC TGT AGT TCC TGC TG-3′, Mcpt5 reverse: 5′-CAG TTG ACA ATC TGG GTC TT-3′; Mcpt6 forward: 5′-CAT TGA TAA TGA CGA GCC TCT CC-3′, Mcpt6 reverse: 5′-CAT CTC CCG TGT AGA GGC CAG-3′; Mitf forward: 5′-AGA TTT GAG ATG CTC ATC CCC-3′, Mitf reverse: 5′-GAT GCG TGA TGT CAT ACT GGA-3′; Srgn forward: 5′-GCA AGG TTA TCC TGC TCG GAG-3′, Srgn reverse: 5′-GGT CAA ACT GTG GTC CCT TCT C-3′; and Chst15 forward: 5′-GGC TTT TCA GGT CAC CTA GCA-3′, Chst15 reverse: 5′-GAC ATT ATG GGT TCC TCG TTG A-3′. All data were derived from five independent experiments and are presented as the mean ± SD.

Protease activity

Tryptase activity was assessed by the chromogenic substrate S-2288 (Chromogenix, Milan, Italy). One half million cells were lysed with 150 μl of lysis buffer (2 M NaCl and 0.5% Triton-X 100 in PBS) for 30 min on ice. Ten microliters of lysate was then mixed with 90 μl of autoclaved water, followed by the addition of 20 μl of the substrate from a stock solution (10 mM of S-2288). The activity was recorded by reading the absorbance changes at 405 nm over 60 min using a microplate reader (Molecular Devices, Sunnyvale, CA). Each measurement was performed in triplicates or quadruplicates, and the results represent the mean ± SD. Each experiment was repeated at least three times using different batches of cells.

Proteoglycan analysis

Heparin and chondroitin sulfate analysis by reverse-phase ion-pair–HPLC was performed as described previously (26) with 5 × 106 BMMCs cultured under normal copper conditions, excess copper (20 μM), or deprived of copper (BCS: 200 μM).

Tryptase staining

For tryptase activity staining, cytospin slides were incubated for 15 min with a solution of 10 mM Z-Gly-Pro-Arg 4-methoxy-2-naphthylamine (Sigma-Aldrich) in 0.5 M Tris-HCl (pH 7.5) and 5 mg/ml Fast Garnet GBC sulfate salt (Sigma-Aldrich). For immunohistochemistry, staining was performed using a monoclonal anti-tryptase Ab, as described previously (27).

IgE receptor cross-linking

Cells were sensitized with IgE anti-DNP at 0.1 μg/ml overnight. After washing with PBS, cells were resuspended in BMMC media and stimulated with DNP–Human Serum Albumin at 0.5 μg/ml for 1 h. Cell pellets and media were separated by centrifugation at 300 × g for 10 min. β-hexosaminidase activity was measured according to protocol (23).

Histamine and β-hexosaminidase assays

Histamine was quantified using an ELISA Kit (DRG Instruments, Marburg, Germany), according to the instructions provided by the manufacturer. β-hexosaminidase activity was measured as previously described (23).

Statistical analysis

Experimental data were analyzed using Microsoft Excel 2010 software. For multiple comparisons, one-way ANOVA with a Bonferroni correction was performed, followed by the Tukey multiple-comparisons test or a two-tailed t test, when appropriate; significance level was set at 5%. At least three independent experiments were performed for each experimental setting, and data are presented as the mean ± SD.

Results

Copper affects mast cell morphology

To investigate the impact of copper on mast cells, we incubated BMMCs with excess concentrations of copper or with a cell membrane nonpermeable copper chelator (BCS) to cause copper deficiency. Excessive copper, up to 100 μM, was not toxic to mast cells. However, a significant loss of viability was seen at copper concentrations of 500–1000 μM (Fig. 1A). BCS was nontoxic to mast cells up to 500 μM (Fig. 1A). To assess the effect of copper on mast cell morphology, we first stained mast cells with May Grünwald/Giemsa, cultured at normal copper status (5 μM), in the presence of excess copper (10 μM), or after copper deprivation (100 μM BCS). As seen in Fig. 1B, mast cells cultured under normal conditions showed typical metachromatic staining of granules. When subjected to copper overload, a modest decrease in metachromatic staining was observed (Fig. 1B, middle panel). More strikingly, a marked increase in metachromatic staining was seen after copper deprivation (Fig. 1B, right panel). In fact, the metachromatic staining seen after copper chelation was clearly stronger than when mast cells were cultured under standard conditions. To further assess the effect of copper status on mast cell morphology, we conducted TEM analysis. As seen in Fig. 1C (left panel), mast cells cultured under standard conditions showed an abundance of granules containing electron-dense and electron-translucent regions After copper deprivation, an increased abundance of electron-dense regions within granules was observed (Fig. 1C, right panel; quantification shown in Fig. 1D), whereas copper overload caused a tendency toward reduced content of electron-dense regions (Fig. 1C, middle panel). Taken together, these findings indicate that alterations in copper levels produce extensive morphological effects on mast cells, suggesting that normal mast cell homeostasis is dependent on the precise regulation of copper levels.

FIGURE 1.
FIGURE 1.

Copper affects mast cell morphology and copper transport mechanisms without reducing viability. (A) BMMCs (1 × 106 cells per milliliter) were cultured for 7 d in the absence or presence of excess copper at the indicated concentrations (left panel) or in the presence of a copper chelator (BCS; right panel) at the indicated concentrations, followed by measurement of cell viability. (B–E) BMMCs (1 × 106 cells per milliliter) were cultured under normal copper conditions, excess copper (20 μM), or were deprived of copper (BCS: 200 μM). (B) Cytospin slides were prepared and stained with May Grünwald/Giemsa. (C and D) TEM analysis was performed. (C) Representative TEM photographs (main photograph: original magnification ×6000). (D) Quantification of TEM images. Data are mean ± SD (n = 5). (E) BMMCs (0.5–1 × 106 cells per milliliter) were cultured under normal copper conditions (Ctrl), excess copper (20 μM), or were deprived of copper (BCS: 200 μM), followed by Western blot analysis for expression of Ctr1 (left panel) and densitometry from three membranes or CoxIV (right panel). Actin was used as loading control. *p ≤ 0.05, **p ≤ 0.01.

Alterations in copper levels affect the copper-transport machinery

To investigate whether overload or deficiency of copper affects the copper import apparatus in mast cells, we assessed the effects on copper transporter 1 (Ctr1), the main copper importer in mammalian cells. As seen in Fig. 1E, copper overload caused a reduction in Ctr1 protein, whereas copper chelation induced an increase in Ctr1. These findings suggest that culture of mast cells under excessive copper or copper chelation changes the intracellular copper homeostasis, forcing the cells to regulate their copper uptake via Ctr1.

We also assessed the effect of copper overload/deficiency on levels of cytochrome c oxidase IV (CoxIV), a mitochondrial protein that is sensitive to changes in intracellular copper levels, such that copper deficiency typically leads to suppressed levels of this protein (28). We found that the addition of copper chelator caused a marked decrease in the levels of this protein, verifying that the cells were copper deprived (Fig. 1E, right panel). In contrast, copper overload did not affect the levels of CoxIV (Fig. 1E, right panel).

Copper overload and deficiency affect mast cell proteoglycans

The metachromatic staining of mast cells is caused by the highly sulfated heparin or chondroitin sulfate glycosaminoglycan side chains of serglycin proteoglycans, which are stored in the mast cell secretory granules (29, 30). Hence, it is conceivable that the effects of copper overload/deficiency on metachromatic staining (see Fig. 1B) are reflected by effects on proteoglycan content and/or the extent of sulfation. To address this, we performed a structural and quantitative analysis of the heparin and chondroitin sulfate proteoglycans in mast cells. These analyses showed that chondroitin sulfate is the dominant proteoglycan species in the mast cell population used for this study (BMMCs; Fig 2A, 2B). In agreement with the increased metachromatic staining seen after copper deprivation (see Fig. 1B), we noted that the addition of copper chelator caused a significant increase in the total content of chondroitin sulfate (Fig. 2B). Specifically, copper deprivation caused an increase in the amount of chondroitin sulfate disaccharides carrying both 6-O- and 4-O-sulfate groups (6S4S) and 4-O-monosulfated disaccharides (4S) (Fig. 2B). Copper chelation also caused a significant increase in the total amount of highly sulfated (trisulfated) heparin disaccharides (NS6S2S), as well as a trend toward increased overall heparin content (Fig. 2A). Moreover, there was a trend toward decreased heparin content after copper overload. When we instead examined the relative content of different disaccharides, neither copper overload nor deprivation affected the disaccharide composition of heparin (Fig. 2C) (i.e., the percentage of each of the disaccharide species was not altered). However, we noted that the copper deprivation caused a significant increase in the proportion of disulfated chondroitin sulfate disaccharides (6S4S), along with a corresponding decrease in the proportion of nonsulfated disaccharide species (OS) (Fig. 2D).

FIGURE 2.
FIGURE 2.

Proteoglycans are affected by the copper status of mast cells. BMMCs (1 × 106 cells per milliliter) were cultured under normal copper conditions (Ctrl), excess copper (20 μM), or were deprived of copper (BCS: 200 μM). Glycosaminoglycan chains were then isolated from the proteoglycans and depolymerized to disaccharides by treatment with chondroitinase ABC and heparinase I, II, and III. Disaccharides were separated by reverse-phase ion-pair–HPLC. Amounts of heparin (A) and chondroitin sulfate (B) disaccharides in glycosaminoglycan samples from BMMCs cultured under normal copper conditions (Ctrl), with excess copper (20 μM), or after copper deprivation (BCS: 200 μM). Percentage of heparin (C) and chondroitin sulfate (D) disaccharides in glycosaminoglycan samples from BMMCs cultured under normal copper conditions, with excess copper (Cu), or after copper deprivation (BCS). Data are mean ± SD (n = 3). Heparin disaccharides: NAc, HexA-GlcNAc; NS, HexA-GlcNSO3−; NS2S, HexA(2-O-SO3−)-GlcNSO3−; NS6S, HexA-GlcNSO3−(6- O-SO3−); NS6S2S, HexA(2-O-SO3−)-GlcNSO3−(6-O-SO3−); 2S, HexA(2-O-SO3−)-GlcNAc; 2S6S, HexA(2-O-SO3−)-GlcNAc(6-O-SO3−); 6S, HexA-GlcNAc(6-O-SO3−). Chondroitin sulfate disaccharides: OS CS/Hya, nonsulfated disaccharides of chondroitin sulfate or hyaluronan origin; 2S, HexA(2-O-SO3−)-GalNAc; 2S4S, HexA(2-O-SO3−)-GalNAc(4-O-SO3−); 2S4S6S, HexA(2-O-SO3−)-GalNAc(4,6-di-O-SO3−); 2S6S, HexA(2-O-SO3−)-GalNAc(6-O-SO3−); 4S, HexA-GalNAc(4-O-SO3−); 4S6S, HexAGalNAc(4,6-di-O-SO3−); 6S, HexA-GalNAc(6-O-SO3−). (E) Total mRNA was prepared from mast cells cultured under differing copper conditions and was analyzed by qPCR for mRNA encoding Srgn, Ndst2, Chst11, and Chst15. Data are mean ± SD (n = 6). *p ≤ 0.05. GalNAc, N-acetyl-galactosamine; GlcNAc, N-acetyl-glucosamine; HexA, hexuronic acid (either glucuronic acid or iduronic acid).

To address whether the effects of copper starvation on stored heparin/chondroitin sulfate were reflected by corresponding effects on the genes involved in their biosynthesis, we used qPCR analysis. However, deprivation of copper had no significant effects on the expression of the genes coding for serglycin (Srgn; core protein of the heparin and chondroitin sulfate proteoglycans present in mast cell granules), N-deacetylase/N-sulfotransferase-2 (Ndst2; key enzyme in the sulfation of heparin), chondroitin 4-O-sulfotransferase-1 (Chst11), or chondroitin-4,6-sulfotransferase (Chst15); the two latter enzymes have key roles in the sulfation of chondroitin sulfate (Fig. 2E).

Copper overload/deficiency affect the expression of tryptase

Because copper overload/deficiency affect the proteoglycan content of mast cells, we considered the possibility that additional granule constituents also can be regulated by copper levels. To address this possibility, we first assessed the effects of copper overload/deficiency on tryptase, a serine protease that is a major constituent of mast cell granules (31). When staining cytospin slides with Fast Garnet, a reagent that detects trypsin-like activity (32), we noted a decrease in tryptase activity after copper overload (Fig. 3A, compare left and middle panels). In contrast, tryptase activity was substantially increased over the levels in control mast cells after copper deprivation (Fig. 3A, right panel). The effects of copper overload/deficiency on tryptase activity were also confirmed by assessing trypsin-like activity in mast cell extracts using a chromogenic substrate (Fig. 3B).

FIGURE 3.
FIGURE 3.

Tryptase expression in mast cells is affected by copper status. BMMCs (1 × 106 cells per milliliter) were cultured under normal copper conditions, excess copper (20 μM), or were deprived of copper (BCS: 200 μM). (A) Cytospin slides were prepared and stained for tryptase activity. Original magnification ×400. (B) Lysates from cells cultured under the different copper conditions were assayed for trypsin-like activity using S-2288. (C) Total mRNA was prepared from BMMCs cultured under differing copper conditions and was analyzed by qPCR for mRNA encoding tryptase (Mcpt6), Cpa3, or chymase (Mcpt4 and Mcpt5). Data are mean ± SD (n = 5–8). *p ≤ 0.05, ***p ≤ 0.001.

Potentially, the impact of copper overload/deficiency on tryptase levels could occur as an effect at the protein level, but we also considered the possibility that copper overload/deficiency affect tryptase gene expression. To address the latter, we used qPCR; this analysis revealed that copper overload suppressed tryptase gene (Mcpt6) expression (Fig. 3C). Conversely, copper deficiency resulted in a marked elevation of Mcpt6 expression in comparison with nontreated cells (Fig. 3C). In contrast, copper overload or deficiency did not affect the expression of Cpa3, Mcpt4 (chymase), or Mcpt5 (chymase) (Fig. 3C). Hence, these data indicate that the effects of copper overload/deficiency on tryptase activity/protein levels are reflected, at least in part, by corresponding alterations in gene-expression levels.

We also assessed whether copper overload/deficiency affect histamine, one of the hallmark constituents of mast cells (3). However, the levels of histamine stored within mast cells were not affected by copper overload/deficiency (Fig. 4A). Notably though, copper overload led to a modest, but significant, increase in histamine recovered in the cell culture medium, indicative of an increased spontaneous histamine release.

FIGURE 4.
FIGURE 4.

The copper status has minimal effects on histamine content and on the ability of mast cells to undergo immunological activation. (A) BMMCs (1 × 106 cells per milliliter) were cultured for 3 d under normal copper conditions, excess copper (50 μM), or were deprived of copper (BCS: 200 μM). Cells were pelleted by centrifugation. The histamine content of the cell pellet (right panel) and cell supernatant (left panel, corresponding to secreted histamine) was quantified by ELISA. Data are mean ± SD (n = 10–15). (B) BMMCs (1 × 106 cells per milliliter) were subjected to IgER cross-linking (IgE + DNP) or were only sensitized (IgE), as described in Materials and Methods. The β-hexosaminidase content of the supernatants was quantified as a measure of the extent of degranulation. The total β-hexosaminidase content of untreated cells was also quantified (total β-hexosaminidase). Results are given as the percentage release of total β-hexosaminidase. Data are presented as mean ± SD (n = 3). *p ≤ 0.05.

To investigate whether mast cell activation is sensitive to copper levels, we subjected mast cells to copper overload or deficiency and then measured their ability to degranulate in response to IgER cross-linking. As seen in Fig. 4B, copper overload or deficiency did not affect the ability of mast cells to degranulate, as assessed by their ability to release β-hexosaminidase.

Copper regulates the levels of Mitf

Next, we sought to unravel the mechanism by which copper overload/deprivation affects tryptase gene (Mcpt6) expression. Previous findings have indicated a major role for the transcription factor Mitf in promoting Mcpt6 expression (33); therefore, we considered the possibility that copper might affect this transcription factor. Indeed, when incubating BMMCs with excess copper, we noted a suppression of Mitf gene expression (Fig. 5A), which was reflected by a reduction in Mitf protein (Fig. 5B). Conversely, copper deficiency induced a significant increase in Mitf gene expression. Hence, changes in copper status cause alterations in the expression of Mitf; thus, it is conceivable that such alterations in Mitf expression can cause downstream effects on Mcpt6 expression.

FIGURE 5.
FIGURE 5.

Mitf expression in mast cells is influenced by the copper status. BMMCs (1 × 106 cells per milliliter) were cultured for 7 d under normal copper conditions, excess copper (20 μM), or were deprived of copper (BCS: 200 μM). Cells were pelleted by centrifugation. (A) Total RNA was prepared from cell pellets and analyzed by qPCR for the content of mRNA representing Mitf. Data are mean ± SD (n = 4 or 5). (B) Protein extracts were prepared and subjected to Western blot analysis using an anti-Mitf Ab detecting several Mitf isoforms. Actin was used as a loading control. The melanoma cell line B6F10 was used as a reference for the melanocyte Mitf (Mel). All Western blot lanes are from the same membrane. *p ≤ 0.05, **p ≤ 0.01.

Copper affects the MAPK pathway

It has been shown that Mitf is closely linked to the MAPK pathway, through the finding that a RAS–RAF–MEK–ERK1/2 axis targets Mitf for proteasomal degradation (34–36). Moreover, it has been demonstrated that copper can affect this axis by promoting MEK1/2-mediated phosphorylation of ERK1/2 (37). Hence, one plausible explanation for the effects on Mcpt6 transcription is that copper affects MEK1/2, leading to enhanced ERK1/2 phosphorylation, in turn promoting Mitf degradation and suppressed Mcpt6 expression. To evaluate this possibility, we incubated mast cells with PD98059, a highly selective in vitro inhibitor of MEK1/2 activation, followed by an assessment of downstream effects. As shown in Fig. 6, MEK1/2 inhibition in copper-overloaded mast cells indeed led to increased expression of Mitf, as seen at the mRNA (Fig. 6A) and protein levels (Fig. 6B). Moreover, MEK1/2 inhibition in copper-overloaded mast cells resulted in enhanced expression of the Mcpt6 gene (Fig. 6A), increased Mcpt6 protein levels (Fig. 6B), and increased levels of tryptase activity (Fig. 6C). PD98059 treatment also enhanced Mcpt6 and Mitf expression, as well as tryptase activity, in cells that were not treated with copper. However, the extent of enhancement was less pronounced than seen for the copper-treated cells.

FIGURE 6.
FIGURE 6.

Mitf and tryptase expression in mast cells is regulated by MAPK signaling. Mast cells (BMMCs; 1 × 106 cells per milliliter) were cultured for 3 d with a MEK1/2 inhibitor (PD; PD98059), excess copper (50 μM), or excess copper together with PD. (A) Total RNA was prepared and analyzed by qPCR for the content of mRNA representing tryptase (Mcpt6) and Mitf. GAPDH was used as a housekeeping gene. Data are mean ± SD (n = 4 or 5). (B) Cells were pelleted and subjected to Western blot analysis using Abs to Mitf, tryptase (Mcpt6), p-ERK1/2, total ERK1/2, and Ctr1. Actin was used as loading control. (C) Cells were analyzed for tryptase activity using a chromogenic substrate (S-2288). Data are presented as mean ± SD (n = 4). (D) Mast cells (1 × 106 cells per milliliter) were cultured for 3 d under normal copper conditions or with excess copper (20 μM), in the absence or presence of MEK1/2 inhibitor (PD98059) as indicated, followed by preparation of cytospin slides and staining with May Grünwald/Giemsa (original magnification ×400). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

As expected, incubation of copper-overloaded mast cells with the MEK1/2 inhibitor resulted in suppressed phosphorylation of ERK1/2, whereas no effects on ERK1/2 total protein levels were seen (Fig. 6B). It was also noted that MEK1/2 inhibition in copper-overloaded cells caused an increase in Ctr1 protein levels (Fig. 6B), possibly due to a compensatory mechanism to enhance copper import into the cells.

In line with the increase in Mitf expression after MEK1/2 inhibition, we noted that mast cells with suppressed MEK1/2 activity stained more intensely with May Grünwald/Giemsa in comparison with untreated cells (Fig. 6D). This was seen, in particular, when comparing the staining properties of cells incubated with excess copper versus cells incubated with excess copper in combination with the MEK1/2 inhibitor (Fig. 6D).

Increased numbers of tryptase-positive mast cells in patients with MEDNIK syndrome

Human subjects with MEDNIK syndrome have a complex clinical phenotype featuring mental retardation, enteropathy, deafness, neuropathy, ichthyosis, and keratopathy (19). MEDNIK patients have low serum copper levels due to a mutation in the AP1S1 gene, which encodes for the σ1A subunit of adaptor protein complex 1. This complex is important for intracellular trafficking of various transmembrane proteins, including two copper-transporting ATPases (ATP7A and APT7B) (38, 39). Based on the hyperallergenic skin phenotype including pruritis and dermatitis, and our data showing that copper affects mouse mast cells, we hypothesized that MEDNIK patients might manifest alterations in mast cell phenotype. To evaluate this prospect, we obtained multiple skin biopsies from a MEDNIK patient and healthy control subjects and stained them for chymase and tryptase content. The total number of mast cells was significantly higher in the MEDNIK patient (Fig. 7A). As expected, chymase-positive (Fig. 7B) and tryptase-positive (Fig. 7C) mast cells were observed in skin of the healthy subjects and in the MEDNIK patient. However, the intensity of chymase and tryptase staining was higher in tissues from the MEDNIK patient. These findings indicate that MEDNIK syndrome can be associated with increased mast cell numbers, as well as higher content of chymase and tryptase in mast cells.

FIGURE 7.
FIGURE 7.

Increased tryptase-positive mast cells in a patient with MEDNIK syndrome. (A) Quantification of mast cell number in normal control and MEDNIK syndrome skin biopsy sections stained with toluidine blue for metachromatic staining. Data are mean ± SD (n = 4). (B) Representative images of normal control and MEDNIK syndrome skin biopsy sections stained for chymase activity. Note that the MEDNIK patient specimen shows more intense staining, consistent with higher chymase content of mast cells, as well as higher overall mast cell numbers. (C) Representative images of normal control and MEDNIK syndrome skin biopsy sections stained for tryptase. Note that the MEDNIK patient specimen shows more intense staining consistent with higher tryptase content of mast cells. Skin biopsies were obtained from skin of healthy volunteers or from a MEDNIK patient. Biopsies were embedded in paraffin and stained for chymase using chloroacetate esterase and tryptase using a mAb (main photographs: original magnification ×400). **p ≤ 0.01.

Discussion

In this article, we provide evidence suggesting that mast cells are dependent on a precise regulation of copper status to maintain proper granule homeostasis. Copper, thereby, emerges as a novel factor that influences mast cell phenotype.

A major finding in this study was that copper deprivation was associated with enhanced metachromatic staining of mast cells. Strong metachromatic staining with cationic dyes, such as May Grünwald/Giemsa and toluidine blue, is a typical feature of mature mast cells and is a result of binding of the respective dyes to the highly anionic proteoglycans that are stored within the mast cell granules (24, 30, 40, 41). Thus, the enhanced metachromatic staining of mast cells seen after copper deprivation suggests an increased maturation of the cells, as manifested by increased synthesis of highly sulfated proteoglycans. Indeed, we demonstrate that copper deprivation leads to a substantial increase in the total levels of chondroitin sulfate proteoglycans in mast cells. It was also notable that copper deficiency resulted in increased anionic charge density of the chondroitin sulfate proteoglycans, as evidenced by a selective increase in disulfated disaccharide species at the expense of nonsulfated variants. However, the increase in proteoglycans was not matched by a corresponding increase in the expression of a number of proteoglycan biosynthesis enzymes.

Another major finding was that tryptase is highly sensitive to alterations in copper status. First, we show that copper overload results in substantially decreased levels of tryptase activity, whereas, on the contrary, copper deficiency resulted in higher levels of tryptase activity than in cells cultured under normal copper conditions. Potentially, alterations in tryptase activity could be a result of direct effects of copper overload/deficiency on the enzymatic properties of tryptase. However, we noted that the alterations in tryptase activity were accompanied by corresponding changes in tryptase gene (Mcpt6) expression, suggesting that copper affects the production of tryptase rather than modulates its enzymatic activity.

The mechanism by which copper affects Mcpt6 gene transcription is intriguing. Previous studies have identified a key role for Mitf in driving the expression of Mcpt6 in mast cells by binding to CACATG and CATCTG motifs in the Mcpt6 promoter (33, 42); therefore, we regarded effects on Mitf as a candidate mechanism for explaining the effects of copper on Mcpt6 transcription. In accordance with this hypothesis, we found that Mitf was sensitive to alterations in copper status. Importantly, the effects on Mitf levels were compatible with the corresponding effects seen on Mcpt6 expression (i.e., copper overload resulted in decreased Mitf expression accompanied by reduced Mcpt6 transcription, whereas copper depletion resulted in increased Mitf and Mcpt6 gene expression). Hence, our data indicate that copper influences Mcpt6 expression by affecting Mitf.

Previous studies have suggested that Mitf, in addition to affecting the expression of Mcpt6, influences the expression of other mast cell–restricted proteases, including the chymases Mcpt4 (43) and Mcpt5 (44). Therefore, it was somewhat unexpected that neither of these latter genes was affected by alterations in copper status. We cannot explain with certainty why altered copper status preferentially affects Mcpt6. One possibility is that the Mcpt6 gene is more sensitive to changes in Mitf levels than are the genes coding for Mcpt4 and Mcpt5 (i.e., the changes in Mitf levels due to copper alterations may be sufficient to affect Mcpt6 but not the other mast cell protease genes). However, this remains to be clarified.

To explain how copper affects Mitf expression, we sought to identify upstream signaling mechanisms that could influence Mitf. It is known that Mitf expression is regulated by MAPK signaling (45); therefore, we focused on this pathway. Intriguingly, there is evidence suggesting that the MAPK pathway can be influenced by copper, through the finding that copper can enhance the MEK1/2-dependent phosphorylation of ERK1/2, leading to enhanced proteasomal degradation of Mitf (37). Thus, a likely scenario would be that inhibition of MEK1/2 results in decreased ERK1/2 phosphorylation, in turn leading to higher levels of Mitf accompanied by enhanced Mcpt6 expression. Accordingly, copper overload would lead to the opposite (i.e., decreased Mitf levels, leading to suppressed Mcpt6 expression). Indeed, our findings support this scenario. Altogether, we may suggest a mechanism whereby copper affects the activity of MEK1/2, leading to downstream effects on ERK1/2, Mitf, and Mcpt6.

Our findings may also have bearing on the understanding of human pathologies associated with altered copper homeostasis. Such pathologies include MEDNIK syndrome, a disease associated with copper deficiency and concurrent ichthyosis and erythroderma, and Menkes disease (20). In this article, we present data suggesting that MEDNIK syndrome can be accompanied by an increase in the number of tryptase-positive mast cells. Based on our findings, it seems conceivable that the copper deficiency in these patients contributes to such effects on mast cells. It should be noted that the MEDNIK patient samples were from a young child (MEDNIK patients have a short life span), whereas healthy control samples were taken from adult subjects (because of ethical considerations); therefore, it cannot be excluded that an age-associated bias might represent a confounding factor. However, arguing against this scenario, it has been reported that the numbers and granular content of mast cells increase with age (46–48). Untreated patients with Menkes disease also show low levels of serum copper; thus, it is possible that mast cells are also affected in this illness. However, this question remains to be investigated, because we did not have access to Menkes patients not already under treatment with copper replacement.

As judged by the data displayed in Fig. 7, it appears that the numbers of mast cells increase under copper deficiency. This may seem to be at odds with the lack of effect of low copper conditions on mast cell proliferation in vitro. At present, we cannot explain this dichotomy. However, one potential explanation could lie in the kinetics and spatial differences between the two settings: the mast cells in the in vitro culture setting are derived within a short time frame (∼3 wk), whereas the MEDNIK sample was from a 2-y-old subject. Further, the cultured mast cells are grown in a suspension culture, whereas the MEDNIK sample mast cells reside in connective tissue, having extensive contacts with extracellular matrix compounds. Hence, it is possible that the effects of copper may be differentially manifested depending on the time of exposure and on tissue location.

Taken together, our findings introduce the possibility that mast cell homeostasis is critically dependent on an adequate copper status, with alterations in copper levels leading to aberrant expression of key genes and effects on mast cell maturation. Hence, our findings suggest that copper may have a hitherto unrecognized role in the regulation of mast cell function. As an extension of these findings, it will be of interest to investigate whether the contribution of mast cells to various pathological settings can be subject to regulation due to changes in copper status.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank Inger Eriksson for expert technical assistance with the proteoglycan analysis.

Footnotes

  • This work was supported by grants from the Swedish Research Council, the Swedish Cancer Foundation, the Swedish Heart-Lung Foundation, the Torsten Söderberg Foundation (all to G.P.), the Magnus Bergvall Foundation, the Åke Wiberg Foundation (both to H.Ö.), and National Institutes of Health Grants Z01 HD008768 and Z01 HD008927 (to S.G.K.).

  • Abbreviations used in this article:

    BCS
    bathocuproinedisulfonic acid disodium salt
    BMMC
    bone marrow–derived mast cell
    CoxIV
    cytochrome c oxidase IV
    CPA3/Cpa3
    carboxypeptidase A3
    Ctr1
    copper transporter 1
    Mitf
    microphthalmia-associated transcription factor
    qPCR
    quantitative real-time PCR
    TEM
    transmission electron microscopy.

  • Received May 31, 2017.
  • Accepted October 16, 2017.

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