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Department of Food, Nutrition & Food Service Management, The University of North Carolina Greensboro, Greensboro, NC 27412-5001
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGMENTS
LITERATURE CITED
ABSTRACT 
Although dietary copper (Cu) deficiency has been associated with decreased production of interleukin-2 (IL-2) by activated splenic mononuclear cells in rodent models, the basis for this relationship and its relevance for humans remain unknown. To address these matters, we have developed an in vitro model of cellular copper deficiency by treating Jurkat, a human T-lymphocyte cell line, with low concentrations of 2,3,2-tetraamine (2,3,2-tet), a high affinity copper chelator. Exposure to 5-20 µmol/L 2,3,2-tet for 35 h decreased cell copper and the activity of Cu,Zn-superoxide dismutase (Cu,Zn-SOD) by 30-40% and IL-2 production by 60-70% in cultures activated with phytohemagglutinin and phorbol myristate acetate. Similarly, IL-2 mRNA levels were 40-70% lower in chelator-treated cells than in untreated cells at 3-12 h after activation. In contrast, chelator treatment had no significant effect on cell viability, growth, protein synthesis or mitochondrial activity. The presence of a slight molar excess of copper, but not zinc or iron, during exposure to 2,3,2-tet prevented the chelator-induced decrease in Cu,Zn-SOD activity and the reductions in IL-2 mRNA and bioactivity. Moreover, binding of diferric transferrin (Tf) and cellular uptake of Tf-59Fe by Jurkat cells were not increased by 2,3,2-tet, indicating that chelator-treated cells were not iron deficient. Finally, incubation of human peripheral blood mononuclear cells (PBMC) with 2,3,2-tet decreased mitogen-induced IL-2 production by 50% compared with untreated controls. These data indicate that a decline in copper status decreases IL-2 production by activated human T-cells due to reduced synthesis and/or stability of IL-2 mRNA.
Key words: Cu deficiency, 2,3,2-tetraamine, interleukin-2, Jurkat human T-cell line, peripheral blood mononuclear cells.INTRODUCTION
Numerous investigators have demonstrated that copper (Cu) deficiency impairs both innate and acquired activities of the immune system in laboratory and domestic animals (Prohaska and Failla 1992
). The general relevance of these findings for humans remains unclear for the following reasons: severe Cu deficiency is rare in humans; there have been few controlled investigations of the impact of clinically diagnosed Cu deficiency on immunocompetence; and the lack of reliable and sensitive indicators of marginal and moderate Cu deficiency has precluded examination of the activities of leukocytic cells from individuals with such conditions. To begin to address the issue of relevance, we have developed an in vitro model of Cu deficiency to study the influence of cellular Cu status on the activities of human T-lymphocytes.
The cytokine interleukin-2 (IL-2)5 is a product of activated T-lymphocytes and has a central role in the regulation of acquired immune responses. We previously reported that the suppressed mitogenic responsiveness of splenic mononuclear cells (MNC) isolated from Cu-deficient rats was highly correlated with reduced interleukin-2 (IL-2) activity in these cultures (Bala and Failla 1992). In vitro addition of exogenous IL-2 increased [3H]-thymidine incorporation into DNA to control levels. Moreover, the addition of physiological levels of Cu, but not Zn or Mn, to cultures of MNC isolated from Cu-deficient rats restored mitogen-stimulated IL-2 production and [3H]-thymidine incorporation into DNA to control levels. The basis for the decreased generation of bioactive IL-2 by splenic MNC isolated from Cu-deficient animals remains unknown.
The cell model selected for the present investigations is Jurkat, a human T-lymphocyte cell line that has been used extensively for studying the regulation of IL-2 gene expression (Schwartz et al. 1993). Jurkat cells do not constitutively produce IL-2, but can be activated to synthesize and secrete this cytokine by exposure to T-cell mitogens and phorbol esters. To decrease cellular Cu status, Jurkat cells are incubated with the high affinity Cu chelator 2,3,2-tetraamine (2,3,2-tet; Fawcett et al. 1980, Zhang et al. 1995). The results demonstrate that treatment of cultures with concentrations as low as 5 µmol/L 2,3,2-tet selectively decreases cellular Cu content, Cu,Zn-superoxide dismutase (Cu,Zn-SOD) activity, IL-2 bioactivity and IL-2 mRNA levels in activated Jurkat cells without significantly altering general cellular activities. Moreover, exposure of cultures of normal human peripheral blood mononuclear cells (PBMC) to 2,3,2-tet also impairs IL-2 production, demonstrating that the effect of the Cu chelator is not limited to the transformed cell line.
MATERIALS AND METHODS
-bis[2-aminoethyl]-1,3-propanediamine; Fisher Scientific, Pittsburgh, PA) was added to culture medium at indicated concentrations from a 20 mmol/L aqueous stock solution. Trace metals were added to culture medium by properly diluting stock solutions containing 10 mmol/L metal (as the chloride salt) in 1.0 mmol/L HCL.
70°C after removal of residual cells by centrifugation at 1000 × g for 15 min at 4°C. IL-2 bioactivity was measured by modification of the CTLL-2 bioassay of Gillis et al. (1978) that has been described in detail and validated previously (Hopkins and Failla 1995). Briefly, supernatants from activated cultures were serially diluted (1:2) in RPMIc to a final dilution of 1:64. Recombinant human IL-2 (Genzyme) was used as the standard. A known quantity was diluted in the same manner as the test samples. Aliquots (100 µL) of diluted samples and standards were added to wells containing 2 × 104 CTLL-2 cells in 100 µL RPMIc. Cultures were incubated for 20 h, 20 µL alamar BlueTM dye (Alamar Biosciences, Sacramento, CA) was added to each well and then cultures were incubated for an additional 20 h before measuring absorbance (570-620 nm). All samples and standards were assayed in triplicate at each dilution. Calculated activities for samples represent the mean value for dilutions having a corrected absorbance that was 50% of the maximum absorbance for the standard.
Northern analyses.
Total RNA was isolated from cells using either Trizol (Life Technologies, Gaithersburg, MD) or column kits (Qiagen, Chatsworth, CA) according to the manufacturers' protocols. RNA was fractionated by electrophoresis in 1.2% agarose, 0.5 mol/L formaldehyde denaturing gels and transferred onto nylon membranes (Hybond N; Amersham, Arlington Heights, IL) by capillary blotting using conventional methods. After fixing RNA to membranes by UV irradiation, membranes were hybridized with PCR-synthesized (Perkin Elmer, Norwalk, CT) cDNA probes labeled with [
-32P]dCTP, (>111 TBq/mol; Amersham). The following primers were used: IL-2 5 primer, 5-ATGTACAGGATGCAACTCCTGTCTT-3 and 3 primer, 5-GTCAGTGTTGAGATGATGCTTTGAC-3, generating a 457-bp fragment (Somoza et al. 1994); and GAPDH 5 primer, 5-CTACTGGCGCTGCCAAGGCTGT-3 and 3 primer, 5-GCCATGAGGTCCACCACCCTGT-3, generating a 358-bp fragment (Benjamin et al. 1994). Signals generated by the probes were assessed by phosphorimaging (Molecular Dynamics, Sunnyvale, CA) and autoradiography.
Biochemical analyses.
To analyze cell Cu content, Jurkat cells were incubated in RPMIc with either 0, 5 or 20 µmol/L 2,3,2-tet. After 35 h, cells were collected, washed three times with Hanks' balanced salt solution and resuspended in deionized water at 1010 cells/L before dilution with an equal volume of 30 mmol/L nitric acid. Dr. Susan Percival, University of Florida, analyzed the Cu content of cells by graphite furnace atomic absorption spectrophotometry as previously described (Percival and Layden-Patrice 1992). Cu,Zn-SOD activity was evaluated in sonicates of cells cultured for 35 h in the absence or presence of the chelator, with or without the simultaneous addition of Cu, Zn or Fe. Enzyme activity was determined by measuring the inhibition of pyrogallol auto-oxidation as described by Prohaska et al. (1983). To assess the effect of treatment with 2,3,2-tet on amino acid uptake and protein synthesis, cultures were exposed to medium with or without chelator for 35 h, washed, and resuspended in fresh medium at 109 cells/L. Aliquots (1 mL) were transferred to 12-well dishes and activated with PHA and PMA as outlined above. After 19 h, 37 kBq [4,5
3H]-leucine (sp. act. 6 TBq/mmol, Amersham) was added to each well and cultures were incubated for 1 h. Cells were collected, washed and sonicated, and the quantities of 3H in the sonicate and the acid-precipitable fraction of sonicate were determined as recently described (Martin et al. 1996). Similarly, mitochondrial activity was determined in activated cultures that had been pretreated with 0, 5 or 20 µmol/L 2,3,2-tet using alamar BlueTM (Alamar Biosciences), a chromogenic substrate that changes color in response to cellular respiration. Chelator-treated and control cells were transferred to 96-well dishes (109 cells/L; 200 µL/well) and activated with PHA and PMA. Dye (20 µL/well) was introduced either at the same time as or 12 h after the addition of the activators. Cultures were incubated at 37°C for an additional 8 h before measuring absorbance (570-620 nm). Transferrin-[59Fe] was prepared according to Klausner et al. (1983) and the binding and uptake of [59Fe] was determined using standard procedures. Cellular protein was determined with a kit (BioRad, Hercules, CA), using the method of Bradford (1976).
Statistical analysis.
All experiments were repeated at least twice using triplicate cultures for each variable. Data were analyzed by Student's t test or ANOVA using the General Linear Models (GLM) procedure followed by Tukey's multiple range test (SAS Institute, Cary, NC). Results are presented as means ± SEM. Differences are considered significant when P < 0.05.
RESULTS
). Initial studies showed that exposure of Jurkat cells to the Cu chelator 2,3,2-tet (20 µmol/L) for 24, 35 and 48 h before activating cells with PHA and PMA decreased IL-2 bioactivity in cultures by 50, 70 and 85%, respectively, compared with that in cultures that were not treated with the chelator (Fig. 1). Longer periods of exposure to 2,3,2-tet (as long as 135 h) did not attenuate IL-2 bioactivity further. In subsequent experiments with Jurkat cells, cultures were incubated in medium containing the Cu chelator for 35 h before activation. Production of IL-2 by activated Jurkat cells was still decreased 70% when the concentration of 2,3,2-tet was reduced to as low as 5 µmol/L (Fig. 2). Increasing the concentration of 2,3,2-tet to 40 µmol/L did not enhance its inhibitory effect on production of IL-2. Moreover, elimination of the chelator from medium during the activation phase did not attenuate the suppressive influence of pretreatment of cells with 2,3,2-tet on the production of IL-2 (data not shown). These results suggest that the decreased production of IL-2 was due to depletion of the cellular pool of metabolically active Cu, and not to an inhibitory effect of the chelator on the activity of the mitogens.
. Bars represent means ± SEM (n = 3). An asterisk above a bar indicates that the mean value for cultures treated with 2,3,2-tet differs significantly (P < 0.05) from that for cultures that were not exposed to chelator. The absence of a standard error bar indicates that the SEM was <3% of the mean.
DISCUSSION ). The reduced IL-2 activity in cultures of 2,3,2-tet-treated Jurkat cells suggested that decreased Cu status attenuated IL-2 gene expression. Northern analyses revealed that IL-2 mRNA levels in activated (for 5 h) Jurkat cells exposed to either 5 or 20 µmol/L 2,3,2-tet were ~50% lower than those in mitogen-stimulated cells that had not been treated with the chelator (Fig. 3a). As expected, IL-2 mRNA was not detected in nonactivated cells cultured with or without chelator. Moreover, the level of IL-2 mRNA was lower in 2,3,2-tet-treated Jurkat cells than in control cells at 3, 6, 9 and 12 h after activation with PHA/PMA, indicating that the decrease in cellular Cu did not simply alter the kinetics of the induction of IL-2 gene expression (Fig. 3b).
Fig. 3.
Northern blot analysis of interleukin-2 (IL-2) mRNA levels in control and chelator-treated Jurkat cells following activation. (a) Cultures were incubated with or without the indicated concentrations of 2,3,2-tetraamine (2,3,2-tet) for 35 h. Cells were then activated by the addition of phytohemagglutinin-P and phorbol myristate acetate (2 mg/L and 10 µg/L, respectively). Total RNA was isolated from 2.5 × 107 cells 5 h after addition of mitogens, and 30 µg/treatment was subjected to electrophoresis, blotting and hybridization with probes specific for human IL-2 and GAPDH mRNAs as described in Materials and Methods. The schematic indicates the levels of IL-2 mRNA relative to GAPDH mRNA. (b) Cultures were incubated with 5 µmol/L 2,3,2-tet for 35 h. Cells were then activated with PHA/PMA, and total RNA was isolated from cells at indicated times following addition of mitogen and analyzed as above. Each panel represents data from a single experiment. Experiments were repeated twice.
[View Larger Versions of these Images (24 + 28K GIF file)]
), the specificity of the impact of the chelator on Jurkat cells was examined. The simultaneous addition of 25 µmol/L Cu and 20 µmol/L 2,3,2-tet to the medium of Jurkat cultures prevented the chelator-induced suppression of cellular Cu (data not shown), Cu,Zn-SOD activity and the decline in IL-2 production (Fig. 4). Similarly, the presence of 7 µmol/L Cu during exposure of cells to 5 µmol/L 2,3,2-tet prevented the decrease in cellular Cu, Cu,Zn-SOD activity and IL-2 mRNA levels (data not shown). In contrast, the addition of supplemental Zn or Fe (25 µmol/L) to medium did not block the suppressive influence of 20 µmol/L 2,3,2-tet on either the production of IL-2 or the activity of Cu,Zn-SOD (Fig. 4).
Fig. 4.
Supplemental Cu, but not Zn or Fe, prevents 2,3,2-tetraamine (2,3,2-tet)-induced reduction in interleukin-2 (IL-2) production and Cu,Zn-superoxide dismutase (Cu,Zn-SOD) activity in Jurkat cells. Cells were cultured in RPMIc alone or supplemented with 25 µmol/L of the indicated trace metal in the presence or absence of 20 µmol/L 2,3,2-tet for 35 h. Cells were then activated as described in the legend for Figure 1. IL-2 activity in culture supernatants and Cu,Zn-SOD activity in cells were assessed as in Figures 1 and 2, respectively. Bars represent means ± SEM (n = 3). An asterisk above a bar indicates that the mean for cultures treated with the chelator differs significantly (P < 0.05) from that for cultures that were not exposed to chelator. The absence of a standard error bar indicates that the SEM was <3% of the mean.
[View Larger Version of this Image (23K GIF file)]
). Binding of diferric [59Fe]-transferrin to control and 2,3,2-tet-treated cells at 4°C was 200 ± 6 and 195 ± 4 pmol/106 cells, respectively (P > 0.05). Accumulation of transferrin-[59Fe] by control and 2,3,2-tet-treated cells was 3.2 ± 0.2 and 2.5 ± 0.4 pmol/106 cells, respectively, after incubation of cultures with [59Fe]-transferrin for 1 h at 37°C (P > 0.05). These data indicate that treatment with 20 µmol/L 2,3,2-tet for 35 h did not reduce cellular Fe to the level required for up-regulation of cell surface transferrin receptors.
Fig. 5.
Exposure to 2,3,2-tetraamine (2,3,2-tet) attenuates the production of interleukin-2 (IL-2) by activated human peripheral blood mononuclear cells (PBMC). Freshly isolated PBMC were cultured in 24-well plates containing RPMIc with or without 20 µmol/L chelator for 24 h. Phytohemagglutinin-P (2 µg/well) was then added to cultures to activate cells; 40 h later, supernatants were collected to measure IL-2 bioactivity as described in Materials and Methods. Data are presented for cells from one representative subject. The asterisk above the error bar indicates that the mean value for triplicate cultures treated with 2,3,2-tet differs significantly (P < 0.05) from that for cultures that were not exposed to chelator.
[View Larger Version of this Image (17K GIF file)]
). Moreover, addition of physiological levels of Cu to cultures of MNC isolated from Cu-deficient rats restored IL-2 production and DNA synthesis to control levels. These data suggested that the defect that resulted in decreased production of IL-2 by cells isolated from Cu-deficient animals resided within the mononuclear cell population itself, although the mechanism responsible for this change remained unknown. Furthermore, the potential relevance of these observations to human health is unclear because severe Cu deficiency is rarely diagnosed in the human population and the impact of Cu deficiency on the production of IL-2 by human cells has not been examined. This led us to develop an in vitro model for studying the impact of low Cu status on the function of human lymphocytes and for exploring the basis of impaired production of IL-2 that is associated with Cu deficiency.
).
). Therefore, additional studies were performed to determine whether 2,3,2-tet selectively reduced cellular Cu status in test cultures. Simultaneous addition of a slight molar excess of Cu, but not Zn or Fe, to culture medium containing 2,3,2-tet blocked the chelator-induced declines in Cu,Zn-SOD activity and IL-2 production. We further examined the possibility that exposure to 2,3,2-tet induced a secondary Fe deficiency in light of recent reports showing that iron uptake by yeast is dependent on the functional integrity of FET3, a Cu-dependent ferroxidase (Askwith et al. 1994, Dancis et al. 1994), and conflicting data in the literature concerning the adverse effect of iron deficiency on IL-2 production (Kemp 1993, Lucas et al. 1995, Omara and Blakley 1994). Cellular Fe deficiency is associated with increased expression of transferrin receptor on the cell surface and increased uptake of Tf-Fe (Leibold and Guo 1992). We found that 2,3,2-tet treatment did not induce a secondary Fe deficiency in our model because the binding of Fe2-Tf and the uptake of Tf-Fe were similar in chelator-treated and control cells.
FOOTNOTES
Manuscript received 5 July 1996. Initial reviews completed 8 August 1996. Revision accepted 17 October 1996.
ACKNOWLEDGMENTS
We are grateful to Sue Percival for quantifying cellular copper by graphite furnace atomic absorption spectrophotometry and to Vivian Bullard for preparation of the manuscript.
LITERATURE CITED
-Carotene and lutein protect HepG2 human liver cells against oxidant-induced damage.
J. Nutr.
1996;
126:2098-2106
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