Copper Deficiency Suppresses Effector Activities of Differentiated U937 Cells1,2

Biochemical and Molecular Action of Nutrients

Copper Deficiency Suppresses Effector Activities of Differentiated U937 Cells1,2 Zhixin L. Huang and Mark L. Failla3 Department of Nutrition and Foodservice Systems, The University of North Carolina at Greensboro, Greensboro, NC 27402

KEY WORDS: ● copper inflammatory mediators

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U937 human promonocytic cells

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respiratory burst

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bactericidal activity

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Inadequate Cu nutriture due to dietary deficiency and inherited diseases in Cu transport impairs both the innate and acquired branches of the immune system (Failla and Hopkins 1998, Prohaska and Failla 1993). Cu deficiency suppresses interleukin-2 (IL-2)4 secretion (Bala and Failla 1992, Hopkins and Failla 1997a) and proliferation of activated T cells (Bala et al. 1991, Lukasewycz et al. 1985), attenuates the respiratory burst and microbicidal activities of phagocytic cells (Babu and Failla 1990a and 1990b) and decreases the number of mature neutrophils in peripheral blood (Percival 1995). Limited studies also indicate that Cu deficiency alters the secretion of inflammatory mediators, such as tumor necrosis factor-␣ (TNF-␣) (Gengelbach et al. 1997), IL-1 (Lukasewycz and Prohaska 1990) and prostaglandin E2 (PGE2) (Iwakiri et al. 1998). The specific roles for Cu in the development, activa-

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ABSTRACT Dietary copper (Cu) deficiency impairs both innate and acquired branches of immunity. Specific roles of Cu in the activation and effector activities of host-defense cells remain largely unknown. The effects of Cu status on effector activities of a monocytic cell line were investigated as an initial step in the elucidation of specific functions of Cu in phagocytic cells. Exposure of differentiating U937 human promonocytic cells to 5 ␮mol/L 2,3,2-tetraamine (tet), a high affinity Cu chelator, for 4 d decreased cellular Cu by 62% without altering cellular Cu,Zn-superoxide dismutase (SOD) activity, Zn content, mitochondrial activity and protein synthesis. In contrast, Cu deficiency suppressed the respiratory burst activity and markedly compromised the ability of U937 cells to kill Salmonella. Similarly, treatment of RAW264.7 murine macrophages with 5 ␮mol/L tet decreased cell Cu by 78% and Cu,Zn-SOD activity by 15% and increased bacterial survival by 180%. The tet-induced impairment of respiratory burst and bactericidal activities was blocked in cultures supplemented with Cu, but not Zn or Fe. In addition, lipopolysaccharide (LPS)-induced secretion of the inflammatory mediators, tumor necrosis factor-␣, interleukin (IL)-1␤, IL-6 and prostaglandin E2 (PGE2), was decreased by 30 – 60% in tet-treated U937 cells. Flow cytometric analysis of the surface antigens CD11b and CD71 showed that the suppressed activities of Cu-deficient cells were not due to an attenuation in the degree of differentiation or secondary iron deficiency. These data demonstrate that U937 cells provide a useful model for examining the biochemical roles of Cu in monocyte activity. J. Nutr. 130: 1536 –1542, 2000.

tion and effector activities of host-defense cells remain largely unknown. The goal of this study was to establish a human phagocytic cell model for the elucidation of functions of Cu in the effector activities of monocytes and macrophages. U937, established by Sundstrom and Nilsson (1976) from histiocytic lymphoma, is a well-characterized human promonocytic cell line. Exposure of these cells to various compounds such as phorbol 12-myristate 13-acetate (PMA), 1,25-dihydroxyvitamin D-3, retinoic acid and interferon-␥ induces differentiation (Caron at al. 1994, Ishizuka et al. 1995, Spittler et al. 1997b) and the associated display of effector activities, such as respiratory burst (Chateau et al. 1996), phagocytosis (Sundstrom and Nilsson 1976), microbicidal killing (Caron et al. 1994) and secretion of cytokines (Ishizuka et al. 1995). Preliminary studies in our laboratory showed that the PMA-induced production of superoxide anion (O2 䡠 ⫺), i.e., respiratory burst activity, by differentiated U937 was greater than that generated by similarly treated cultures of differentiated THP1 (another human promonocytic cell line) and the murine macrophage-like cell lines J774A and RAW264.7. This enhanced responsiveness of U937 resulted in its selection for the studies described below. Cu deficiency was induced by treatment of the cells with 2,3,2-tetraamine (tet), a high affinity Cu chelator (Fawcett et al. 1980). This chelator has been used successfully to induce Cu deficiency in several other cell lines (Hopkins and Failla 1997a, Zhang et al. 1995). Our

1 Presented in part at Experimental Biology 99, April 1999, Washington, DC [Huang, Z. & Failla, M. L. (1999) Copper deficiency impairs respiratory burst, killing activity and TNF-␣ secretion in human U937 cells. FASEB J. 13: A372 (abs.)]. 2 Supported in part by U.S. Department of Agriculture NRI 9803775 and NC Institute of Nutrition. 3 To whom correspondence should be addressed. 4 Abbreviations used: Cu,Zn-SOD, copper,zinc-superoxide dismutase; Ig, immunoglobulin; IL, interleukin; LPS, lipopolysaccharide; MFI, mean fluorescence intensity; NF, nuclear factor; O2 䡠 ⫺, superoxide anion; PGE2, prostaglandin E2; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; ROS, reactive oxygen species; tet, 2,3,2-tetraamine; TNF-␣, tumor necrosis factor-␣.

0022-3166/00 $3.00 © 2000 American Society for Nutritional Sciences. Manuscript received 9 November 1999. Initial review completed 28 December 1999. Revision accepted 16 February 2000. 1536

COPPER AND EFFECTOR ACTIVITIES OF MONOCYTIC CELLS

results demonstrate that exposure of differentiated cultures of U937 cells to a low concentration of tet selectively decreases cellular Cu status without altering metabolic integrity and that the Cu deficiency induced suppresses respiratory burst and bactericidal activities and lipopolysaccharide (LPS)-mediated secretion of inflammatory mediators. MATERIALS AND METHODS

supernatant were added to wells containing 150 ␮L of 50 mmol/L Tris-HCl and 1 mmol/L diethylenetriamine pentaacetic acid, pH 8.4. The reaction was initiated by adding 50 ␮L of 0.4 mmol/L pyrogallol in 10 mmol/L HCl, and the absorbance at 320 nm was monitored every 10 s for 2 min at room temperature in the microplate reader (PowerWavex, Bio-Tek Instruments, Winooski, VT). Cu,Zn-SOD activity of sample was determined by comparing the extent of inhibition of the rate of pyrogallol autoxidation with that of a known quantity of purified Cu,Zn-SOD from bovine erythrocytes. One unit of SOD standard inhibited the rate of pyrogallol autoxidation by 50%. Cellular protein was determined by bicinchoninic acid assay (Pierce, Rockford, IL). Details concerning determination of cellular [3H]leucine uptake and incorporation into protein, as well as the assessment of mitochondrial activity using the reduction of Alamar Blue dye, have been described elsewhere (Garrett et al. 1999, Martin et al. 1996). Respiratory burst activity. PMA-stimulated production of superoxide anion (O2 䡠 ⫺) was measured by a slight modification of the SOD-inhibited reduction of ferricytochrome c described by Babu and Failla (1990b). Monolayers of test cells were washed with PBS (pH 7.4) before the addition of the following to each well: 0.25 mL of RPMI 1640 without phenol red; 0.50 mL PBS containing 2 ␮mol/L PMA and 100 ␮mol/L horse heart ferricytochrome c; and 0.25 mL PBS with or without 300 U bovine erythrocyte SOD. The plates were incubated at 37°C for 60 min and placed on ice to stop the reaction. Samples were transferred to microfuge tubes and centrifuged at 5000 ⫻ g and 8°C for 10 min. Absorbance (A) of the supernatants was measured at 550 and 675 nm to obtain a corrected absorbance (A550 ⫺ A675). After subtracting the corrected absorbance of samples with added SOD from those without the enzyme, O2 䡠 ⫺ production was calculated using the molar extinction coefficient (E1 cm ⫽ 21 ⫻ 103 L/(mol䡠cm) for ferricytochrome c and reported as nmol O2 䡠 ⫺ generated/mg cellular protein. Bactericidal activity. The killing of Salmonella typhimurium was determined by a modification of the fluorescence microplate assay of Shiloh et al. (1997). Approximately 10 h before testing control and tet-treated (5 ␮mol/L) cultures of U937 and RAW264.7 cells, S. typhimurium was inoculated into LB broth (DIFCO, Detroit, MI) at 37°C with shaking at 200 rpm. An aliquot of log-phase bacterial culture (1–10 ⫻ 1011/L) was diluted to 2.5 ⫻ 109/L in RPMI containing 10% human serum and incubated at 37°C for 30 min for opsonization. Afterward, 100 ␮L of RPMI without antibiotics and 50 ␮L (1 ⫻ 105) opsonized S. typhimurium were added to each well. Before incubation at 37°C, the experimental plate was centrifuged at 250 ⫻ g for 5 min to enhance the contact between S. typhimurium and the cells. After 60 min, 25 ␮L of 17 mmol/L sodium deoxycholate was added to wells, and samples were pipetted to disrupt U937 cells. Then, 25 ␮L of Alamar Blue dye in basal RPMI (4:1) was added to wells and the plate was returned to the incubator for an additional 2 h. Fluorescence intensity in each well was measured at excitation (Ex) and emission (Em) wavelengths of 530 and 590 nm, respectively. Wells without bacteria but with the same quantities of reagents were read as background. Pilot studies showed that fluorescent intensity from the reduction of Alamar Blue was directly proportional to the number of live bacteria in wells. Phagocytic activity. Phagocytosis of fluorescein-labeled E. coli was determined with a slight modification of the protocol provided by Molecular Probes. Spent medium was removed from control and tet-treated (5 ␮mol/L) differentiated cultures of U937 cells (1 ⫻ 105/ well) in a 96-well plate. Adherent cells were washed with PBS before 100 ␮L fresh RPMI and 50 ␮L opsonized fluorescein-labeled E. coli (1 g modified bacteria/L RPMI containing 100 mL/L human serum) were added to each well. The plate was incubated at 37°C. After 60 min, the medium was removed and 100 ␮L of 2 mmol/L trypan blue was added to all wells to quench extracellular fluorescence. After trypan blue solution was aspirated, fluorescence was monitored at Ex ⫽ 480 nm and Em ⫽ 520 nm. Monolayers that were not exposed to E. coli were handled identically to determine background. Phagocytic activity represents the corrected fluorescence of cultures. Levels of cytokines and PGE2 in medium. LPS (1 mg/L) was added to differentiation medium to activate differentiated U937 cultures. After 24 h, medium was collected from wells, centrifuged

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Materials. Chemicals and tissue culture supplies were purchased from Sigma Chemical (St. Louis, MO) and Fisher Scientific (Pittsburgh, PA) unless otherwise indicated. Alamar Blue was obtained from TREK Diagnostic Systems (Westlake, OH). L-[4,5-3H]Leucine (6.3 TBq Ci/mmol) was purchased from Amersham (Arlington Heights, IL). Fluorescein-labeled Escherichia coli was obtained from Molecular Probes (Eugene, OR). Cell culture and experimental design. U937 human promonocytic cells (ATCC, Rockville, MD) were maintained at 1–10 ⫻ 108/L in Roswell Park Memorial Institute 1640 (RPMI) medium containing 100 mL/L fetal bovine serum, 25 mmol/L glucose, 10 mmol/L HEPES, 22 mmol/L sodium bicarbonate, 4 mmol/L glutamine, 1 mmol/L sodium pyruvate, 100 U/L penicillin, 69 ␮mol/L streptomycin and 0.54 ␮mol/L amphotericin B. To assess the effect of Cu deficiency on the effector activities of monocytes and macrophages, differentiation of U937 cells (5 ⫻ 108/L) in 12- and 96-well flat-bottom plates was induced by the addition of 10 nmol/L PMA. After 2 d, either 5 or 10 ␮mol/L tet was added to fresh medium to induce Cu deficiency. Cultures were maintained in differentiation medium with or without tet for an additional 4 d. Pilot studies showed that cells exhibited maximum respiratory burst activity 6 d after PMA addition and that treatment with tet for 4 d, but not 2 d, decreased superoxide anion (O2 䡠 ⫺) production (see below). Fresh medium was added to cultures every 2 d. Effector activities of U937 cells were examined at the end of the culture period (6 d). When indicated, the medium was supplemented with a trace metal salt to examine the specificity of chelator-induced changes. Equimolar concentrations of tet and either Cu, Zn or Fe were added to such media. Stock solutions (10 mmol/L) of Cu, Zn and Fe were freshly prepared from their chloride salts dissolved in 1 mmol/L HCl. The FeCl3 solution also contained 100 mmol/L sodium ascorbate to facilitate the solubility and cellular uptake of Fe (Han et al. 1995). Appropriate vehicles were added to control media. RAW264.7 murine macrophage-like cells (ATCC) were maintained at 1–10 ⫻ 108/L in Dulbecco’s modified Eagle’s medium containing 25 mmol/L glucose, 10 mmol/L HEPES, 44 mmol/L sodium bicarbonate, 6 mmol/L glutamine, 1 mmol/L sodium pyruvate and antibiotics. Pilot studies showed that addition of 5 ␮mol/L tet to medium containing 2–2.5 ⫻ 108 RAW264.7 cells/L for 2 d decreased cell Cu and bactericidal activity (see below). Fresh medium was provided daily. Cu status and metabolic integrity of phagocytic cell lines. The effect of tet treatment on cell Cu was evaluated by quantifying cellular content of Cu and the activity of copper,zinc-superoxide dismutase (Cu,Zn-SOD) 6 d after induction of differentiation in U937 cells and 2 d after exposure of RAW264.7 cells to medium without or with tet. Cellular Cu and Zn were measured on sonicates by graphite furnace atomic absorption spectrophotometry as described by Reeves et al. (1998). Briefly, cultures of U937 and RAW264.7 cells in T75 flasks were washed twice with PBS before collecting in 2 mL PBS and transferring to a 5-mL centrifuge tube. Each flask was washed with an additional 2 mL PBS that was also added to the collection tube. The cells were pelleted by centrifugation at 250 ⫻ g for 10 min at room temperature and resuspended in 1 mL PBS before sonicating twice (Sonic Dismembrator, Model 60, Fisher Scientific) for 6 s at a setting of 3. Aliquots were added to an equivalent volume of 3.2 mol/L nitric acid and incubated overnight at 37°C before metal analysis. Internal and external standards also were analyzed to ensure accuracy. Cu,Zn-SOD activity was determined by measuring the ability of aliquots of cell extracts to inhibit pyrogallol autoxidation as described elsewhere (Babu and Failla 1990a), except that the assay was modified for use of a microplate reader as follows. Aliquots (100 ␮L) of aqueous

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RESULTS Effects of tet treatment on cellular Cu status and metabolic activities. Exposure of differentiating cultures of U937 to the high affinity Cu chelator tet (5 ␮mol/L) for 4 d decreased Cu content by 62% (P ⬍ 0.05), but did not significantly (P ⬎ 0.05) alter cellular Zn content (Table 1). Surprisingly, the tet-induced reduction in cellular Cu content was not associated with a decline in the activity of Cu,Zn-SOD (Table 1). This resilience of Cu,Zn-SOD activity in differentiated cells treated with tet was specific because exposure of replicating U937 cells to 5 ␮mol/L tet for 2 d significantly reduced cellular Cu content and Cu,Zn-SOD activity by 73.7 ⫾ 2.8% (P ⬍ 0.05; n ⫽ 3) and 30.3 ⫾ 1.8% (P ⬍ 0.05; n ⫽ 6), respectively. Treatment of cultures with PMA for 6 d resulted in the adherence of cells to the vessel surface. Exposure of PMAtreated U937 cells to either 5 or 10 ␮mol/L tet for the final 4 d of the differentiation period did not alter cell adhesion (693 ⫾ 5 and 686 ⫾ 12 ␮g protein/well for control and tet-treated cultures, respectively, P ⬎ 0.05; n ⫽ 3). Protein synthesis and mitochondrial activity were monitored to determine whether tet-induced Cu deficiency affected the general metabolic integrity of U937 cells. Exposure of PMA-treated cells to 10 ␮mol/L tet for 4 d did not compromise either the cellular uptake of [3H]leucine or the incorporation of the amino acid into protein (Table 1). Similarly, mitochondrial activity did not differ in control and Cu-deficient U937 cells as assessed by the rate of reduction of Alamar Blue, a redox-sensitive dye

TABLE 1 Characteristics of differentiated U937 cells exposed to 2,3,2-tetraamine (tet) for 4 d1

Cell metal, nmol/mg protein Cu Zn Cu, Zn-SOD, U/mg protein 5 ␮mol/L tet 10 ␮mol/L tet Metabolic activities [3H]Leu uptake, dpm/(min 䡠 mg protein) [3H]Leu incorporated into protein,2 % Mitochondrial activity,3 units/ (min 䡠 mg protein)

n

Control

⫹Tet

3 3

0.36 ⫾ 0.02 0.84 ⫾ 0.04

0.14 ⫾ 0.02* 1.08 ⫾ 0.08

6 6

5.79 ⫾ 0.11 5.44 ⫾ 0.09

5.82 ⫾ 0.14 5.49 ⫾ 0.11

6

314 ⫾ 9

336 ⫾ 7

6

15.9 ⫾ 0.6

14.2 ⫾ 0.6

6

2.30 ⫾ 0.11

2.26 ⫾ 0.04

1 Values are means ⫾ SEM. Experiments were conducted as described in Materials and Methods. Medium contained 0 vs. 5 ␮mol/L tet for determination of cellular content of Cu and Zn and 0 vs. 10 ␮mol/L for the experiments assessing metabolic activities. The presence of the asterisk as superscript indicates that the mean values for tet-treated cells differed significantly from control (P ⬍ 0.05). 2 Percentage of [3H]leucine incorporated into protein represents the quantity of [3H] in acid-precipitated pellet divided by total cellular [3H]. 3 Arbitrary fluorescence units from the reduction of Alamar Blue by cells.

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(600 ⫻ g for 5 min) and the supernatant was stored at ⫺70°C. Previous studies have shown that the concentrations of proinflammatory cytokines in medium of LPS-treated cultures continue to increase for ⬎24 h (Wang and Alpert 1995). The quantities of TNF-␣, IL-1␤ and IL-6 were determined using ELISA kits (CTYImmune Sciences, College Park, MD), and PGE2 was measured using an EIA kit (Amersham, Piscataway, NJ) according to directions provided by manufacturers. Flow cytometry. Anti-CD11b conjugated with phycoerythin and anti-CD71 conjugated with fluoresceinisothiocyanate were purchased from Pharmingen (San Diego, CA). The negative controls for CD11b and CD71 were antibodies of mouse isotypes immunoglobulin (Ig)G1,␬ and IgG2a,␬, respectively. Spent media were removed from differentiated monolayers of U937 cells before washing with PBS. Cultures were incubated with PBS at 37°C for 45 min and scraped lightly with a rubber policeman to release cells from the dish surface. The suspension was pipetted several times with a narrow-bore tip to break clumps before transferring to 12 ⫻ 75 mm test tubes. Cells were collected by centrifugation (250 ⫻ g for 5 min), washed with PBS containing 10 mL/L fetal bovine serum and 15 mmol/L NaN3, and resuspended in 100 ␮L of the same solution. Aliquots (15 ␮L) of antibodies at concentrations formulated by the manufacturer were added to tubes, and suspensions were incubated in the dark at room temperature. After 30 min, cells were collected, washed and resuspended in 500 ␮L of PBS containing 10 mL/L of fetal bovine serum and 0.33 mol/L formaldehyde. A total of 1 ⫻ 104 cells per sample were analyzed within 2 d after preparation by FACSCalibur (Becton Dickinson, San Jose, CA). Positive cells were defined as those with fluorescence greater than that of the isotypes, and mean fluorescence indices indicated the quantities of antibodies specifically bound to CD11b and CD71. Cell size and granularity were assessed by measuring mean forward scattering and mean side scattering, respectively, for 1 ⫻ 104 cells per sample. Cell cycle analysis was determined using propidium iodide– stained cells as described by Spittler et al. (1997a). Statistical analysis. Unless indicated otherwise, experiments were repeated twice using three independent preparations per treatment for each experiment. Data were analyzed by ANOVA (Excel, Microsoft, Redmond, WA) to determine significant differences (P ⱕ 0.05) and expressed as means ⫾ SEM

(Table 1). These data suggest that tet treatment induced a Cu-deficient state without altering general cellular activities. Effects of Cu deficiency on effector activities. PMAinduced respiratory burst activity, phagocytosis, killing of ingested bacteria and secretion of inflammatory mediators in response to LPS were examined in control and tet-treated cultures of U937 cells. Differentiated cultures of U937 cells generated O2 䡠 ⫺ when treated with 1 ␮mol/L PMA. Cu deficiency suppressed the PMA-induced production of O2 䡠 ⫺ by 22% (Fig. 1A). The attenuated respiratory burst was associated with an impaired ability of Cu-deficient cells to kill S. typhimurium. Bacterial survival in cultures of Cu-deficient U937 cells was 113% (P ⬍ 0.05) greater than that in control cultures (Fig. 1B). The intensity of the fluorescence from the E. coli internalized by control and Cu-deficient cultures of U937 cells was identical (134 ⫾ 5 and 134 ⫾ 2 arbitrary units, respectively; P ⬎ 0.05, n ⫽ 6), indicating that the impaired killing was not associated with a defect in phagocytic activity. To determine whether this adverse effect of tet treatment on U937 cells represented a more general effect of Cu deficiency on phagocytes, Cu deficiency was induced in RAW264.7 murine macrophages. Tet treatment decreased both Cu content (0.07 ⫾ 0.01 vs. 0.02 ⫾ 0.00 nmol/mg protein; P ⬍ 0.05, n ⫽ 3) and Cu,Zn-SOD activity (4.64 ⫾ 0.26 vs. 3.98 ⫾ 0.12 U/mg protein; P ⬍ 0.05, n ⫽ 12), but did not significantly alter cellular Zn content (1.91 ⫾ 0.06 vs. 1.58 ⫾ 0.08 nmol/mg protein for control and tet-treated cells, respectively; P ⬎ 0.05, n ⫽ 3). Similarly, the ability of the Cu-deficient RAW264.7 cells to kill S. typhimurium was significantly (P ⬍ 0.05) impaired, as indicated by the 180% higher number of bacteria in cultures with tet-treated cells compared with the control cultures. Because the use of the chelator to induce Cu deficiency has the potential to alter cellular levels of other essential trace metals, we examined whether the tet-induced changes in cell activities could be blocked by the addition of Cu, Zn or Fe to medium. Respiratory burst activity and bacterial killing capac-

COPPER AND EFFECTOR ACTIVITIES OF MONOCYTIC CELLS

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FIGURE 2 Lipopolysaccharide (LPS)-induced secretion of tumor necrosis factor-␣ (TNF-␣) is decreased in Cu-deficient U937 cells. Cu deficiency was induced by exposure of cultures of U937 cells to 5 ␮mol/L 2,3,2-tetraamine (tet). Secretion of TNF-␣ was stimulated by exposing the cells to 0.1–10 mg/L LPS for 24 h. Data are means ⫾ SEM; n ⫽ 3 cultures at each concentration of LPS. The presence of the asterisk (*) above or below the error bar indicates that TNF-␣ secretion by tet-treated cells was significantly lower (P ⬍ 0.05) than that by control cells treated with the same concentration of LPS.

ity were maintained at the levels of Cu-adequate controls when equimolar concentrations of tet and Cu, but not tet plus either Zn or Fe, were added to medium (Fig. 1A, B). Cu status and secretion of inflammatory mediators. Differentiated cultures of U937 cells constitutively secreted a low level of TNF-␣ (Fig. 2). Medium levels of TNF-␣ increased (P ⬍ 0.05) when cells were exposed to LPS. Cu deficiency depressed both the constitutive and LPS-induced secretion of TNF-␣ by ⬃30% (P ⬍ 0.05). Similarly, Cu deficiency inhibited (P ⬍ 0.05) the secretion of IL-1␤, IL-6 and PGE2 by LPS-treated U937 cells by 23, 58 and 62%, respectively (Table 2). Effects of Cu deficiency on expression of CD11b and CD71. We wished to determine whether tet treatment suppressed effector activities of U937 cells directly by inducing Cu deficiency or indirectly by either suppressing the differentiation process or inducing secondary Fe deficiency. This issue was addressed by examining the expression of CD11b and CD71 on the surface of control and tet-treated U937 cells by flow cytometry. CD11b is the CR3 complement receptor that is highly expressed on monocytes (Farokhzad et al. 1996, Janeway and Travers 1997), whereas CD71 is the transferrin receptor which is up-regulated during Fe deficiency (Leibold and Guo 1992). Both CD11b and CD71 were detected on replicating U937 cells. The percentage of positively stained replicating cells and the mean level of expression per cell (MFI, mean fluorescence intensity) were 41.8 ⫾ 0.8% and 18.4 ⫾ 0.3 MFI, respectively (n ⫽ 3), for CD11b and 49.2 ⫾ 2.9% and 14.2 ⫾ 0.2 MFI, respectively (n ⫽ 3), for CD71.

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FIGURE 1 Cu deficiency suppresses respiratory burst activity and increases bacterial survival in differentiated U937 cultures. Differentiating cultures of U937 cells were treated with either 10 (panel A) or 5 ␮mol/L (panel B) 2,3,2-tetraamine (tet) for 4 d. Replicate cultures with tet contained either no supplemental trace metal or equivalent concentrations of tet and indicated trace metal. Respiratory burst activity and survival of Salmonella typhimurium were determined as described in Materials and Methods. Data are means ⫾ SEM, n ⫽ 6/treatment. Different letters above error bars indicate significant differences (P ⬍ 0.05).

PMA-induced differentiation significantly increased (P ⬍ 0.05) both the percentage of CD11b positive cells and its mean level of expression (Fig. 3A, B). In contrast, the number of cells expressing the CD71 marker was decreased (P ⬍ 0.05) to 3% of the population by 6 d after treatment with PMA (Fig. 3A). Cu deficiency did not significantly alter the profile of either CD11b or CD71 expression on PMA-differentiated U937 cells. Finally, the size, granularity and percentages of cells in G1/G0, S and G2/M phases of the cell cycle did not differ (P ⬎ 0.05) in control and tet-treated differentiated U937 cells (data not shown). The above data demonstrate that Cu deficiency did not impair the PMA-induced maturation of U937 cells or induce secondary Fe deficiency. Thus, suppressed effector functions of tet-treated U937 appear to be related directly to Cu deficiency. DISCUSSION

Previous studies have shown that Cu deficiency impairs the respiratory burst and microbicidal activities of phagocytic cells from humans and animals (Percival 1995, Prohaska and Failla 1993). The results of the present studies demonstrate that exposure of differentiating human U937 cells to low concenTABLE 2 Secretion of interleukin-1␤ (IL-1␤), IL-6 and prostaglandin E2 (PGE2) by lipopolysacchraride (LPS)-treated U937 cells is impaired by Cu deficiency1,2 Inflammatory mediator

⫹10 ␮mol/L tet

Control ng/L

IL-1␤ IL-6 PGE2

210 ⫾ 11 5920 ⫾ 71 1971 ⫾ 120

161 ⫾ 6* 2498 ⫾ 340* 753 ⫾ 27*

1 Values are mean ⫾ SEM for n ⫽ 6 per treatment. The presence of an asterisk as superscript indicates that the response of 2,3,2-tetraamine (tet)-treated cells differed significantly from controls (P ⬍ 0.05). 2 The concentrations of the inflammatory mediators in medium were determined 24 h after the addition of 1 mg/L LPS to medium.

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trations of the Cu chelating agent tet suppressed the same effector activities and decreased the secretion of several proinflammatory cytokines and PGE2 in response to treatment with LPS. The simple interpretation of these observations is that tet-mediated reduction of cellular Cu content impaired the responsiveness of U937 cells to activating stimuli. Results from a series of experiments were obtained in support of this conclusion. Although tet has much greater affinity for Cu than for other transition metals (Fawcett et al. 1980), it was essential to show that the cell content of Cu, but not other essential trace metals, was selectively decreased. Zn and Fe are also essential for various activities of phagocytic cells (Brock 1993, Shankar and Prasad 1998). Direct analysis of Cu and Zn showed that exposure of cells to 5 ␮mol/L tet decreased Cu by 62% without significantly modifying cell Zn (Table 1). Cell Fe content is inversely correlated with the stability of transferrin receptor mRNA and the synthesis of this protein (Eisenstein and Blemings 1998). Therefore, the expression of the transferrin receptor on the surface of cells was measured as an indicator of Fe status. Transferrin receptor levels were not increased in tettreated cells (Fig. 3), indicating that the degree of Cu deficiency did not exceed the threshold associated with the induction of secondary Fe deficiency. Also, tet-mediated alterations in effector activities were blocked when medium was supplemented with Cu, but not Zn or Fe (Fig. 1). Cudeficient cells maintained standard morphologic features of adherent U937 and normal uptake and incorporation of radiolabeled leucine into cell protein (Table 1). Cu is required for the activity of cytochrome c oxidase, and severe Cu deficiency has been shown to cause abnormalities in mitochon-

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FIGURE 3 Cu deficiency does not alter the expression of CD11b and CD71 surface antigens by differentiated U937 cells. U937 cells were differentiated by treatment with 10 nmol/L phorbol 12-myristate 13-acetate (PMA) for 6 d. Cu deficiency was induced by exposing cells to 10 ␮mol/L 2,3,2-tetraamine (tet) from 2 to 6 d of the differentiation process. The percentage of cells expressing the phenotypic marker (% positive cells) shown in panel A and the mean level of marker expression per cell (MFI, mean fluorescence intensity) shown in panel B were determined by flow cytometric analysis of cells stained with monoclonal antibodies as described in Materials and Methods. The mean values for both markers in control and tet-treated cells (n ⫽ 3/treatment) did not differ (P ⬍ 0.05).

drial structure and activity (Bode et al. 1992, Prohaska 1990). We assessed mitochondrial activity in intact cells by monitoring the rate of reduction of Alamar Blue dye. Control and Cu-deficient cells exhibited similar rates of dye reduction (Table 1). Together, these data show that tet selectively decreased cell Cu without compromising general metabolic integrity. We were surprised that the decline in cellular Cu was not associated with a reduction in the activity of Cu,Zn-SOD of differentiated U937 (Table 1). Cu,Zn-SOD activity is often used as a marker of Cu status for isolated cells and animal tissues because it generally decreases in Cu deficiency (Prohaska 1990, Uauy et al. 1985). There are several possibilities for the resilience of Cu,Zn-SOD activity in Cu-deficient differentiated U937 cells. Lost Cu likely comes from the metabolically active pool in which Cu is bound loosely to glutathione (Harris 1997). The Cu associated with cuproenzymes is tightly bound and may not be released until such proteins are degraded. The turnover of Cu,Zn-SOD may be relatively slow in this cell line. Alternatively, Cu,Zn-SOD may have high priority for intracellular Cu in phagocytic cells because they generate large quantities of reactive oxygen species (ROS) for killing microorganisms. Chung et al. (1988) showed that the decline in Cu,Zn-SOD activity in the liver of Cu-deficient rats was much less than the reduction of Cu in this tissue. It is also interesting to note that Cu,Zn-SOD activity was 53% higher (P ⬍ 0.05) in differentiated U937 cells compared with that in replicating, nondifferentiated cultures of U937 (5.79 ⫾ 0.11 vs. 3.78 ⫾ 0.08 U/mg protein, respectively; n ⫽ 6). Despite normal Cu,Zn-SOD activity, the respiratory burst activity was somewhat suppressed in Cu-deficient cultures of differentiated U937 cells compared with Cu-adequate controls. The conversion of O2 to O2 䡠 ⫺ that characterizes the respiratory burst is catalyzed by the protein kinase C (PKC)activated, membrane-bound enzyme NADPH oxidase (Thelen et al. 1993). The O2 䡠 ⫺ generated at or near the cell surface is either released to the extracellular space or confined within phagocytic vacuoles and lysosomes. The impaired microbicidal activity of the Cu-deficient U937 cells (present study) and rat phagocytes (Babu and Failla 1990a and 1990b) suggests that the intracellular levels of ROS also were probably lower in these cells. Cu deficiency may influence the activation of NADPH oxidase by modulating PKC activity. Indeed, Cu deficiency impaired thrombin-mediated induction of PKC activity in rat platelets (Johnson and Dufault 1991) and inhibited mobilization of intracellular calcium required for the translocation of conventional PKC isoforms from cytoplasm to the membrane (Johnson and Dufault 1993). The effect of Cu deficiency on the levels and cellular distribution of PKC in U937 cells is being investigated. The generation of O2 䡠 ⫺ and other ROS represents only one of the factors used by monocytes and macrophages to inactivate invading microorganisms. Others include secreted inflammatory mediators, hydrolytic enzymes and antimicrobial peptides such as defensins (Janeway and Travers 1997). Microbicidal activity of U937 and RAW264.7 cells was more sensitive to cellular Cu status than respiratory burst activity. The possibility that low Cu status adversely influences components in addition to ROS that participate in the killing of microorganisms merits future consideration. Our initial screening of the effects of Cu deficiency on the secretion of several inflammatory mediators represents one of the novel aspects of this study. Cu deficiency suppressed the secretion of the proinflammatory cytokines TNF-␣, IL-1␤ and IL-6 and a major metabolite of arachidonic acid, viz., PGE2, by LPS-treated U937 cells (Table 2). Lower levels of plasma

COPPER AND EFFECTOR ACTIVITIES OF MONOCYTIC CELLS

ACKNOWLEDGMENTS We are grateful to Philip G. Reeves, USDA Grand Forks Human Nutrition Research Center, for quantifying cellular Cu and Zn, Robin

G. Hopkins for helpful discussions and Cissy Geigerman for assistance with flow cytometry.

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TNF-␣ were reported recently in Mo-induced Cu-deficient calves after inoculation with infectious bovine rhinotracheitis virus (Gengelbach et al. 1997). In contrast with our results, LPS-treated splenic macrophages from Cu-deficient mice were reported to secrete higher levels of IL-1 than Cu-adequate cells (Lukasewycz and Prohaska 1990). Also, LPS-induced PGE2 secretion was reported to be increased by thioglycollate-elicited peritoneal macrophages (Iwakiri et al. 1998), but not resident peritoneal macrophages (Koller et al. 1987), from Cu-deficient rats. Differences in cell type, nature of stimulant and the time at which responsiveness to signal was examined are likely to affect outcome for studies addressing the influence of Cu status on secretion of inflammatory mediators. In addition, the in vitro model showed that the decline in cell Cu content directly suppressed effector activities of stimulated U937 cells, whereas responses of phagocytic cells isolated from Cu-deficient animals likely represent the effect of Cu deficiency on phagocytic cells and other cell types whose secretory products modulate phagocytic cell metabolism. How might Cu status influence the secretion of the inflammatory mediators? Generally, the proinflammatory cytokines TNF-␣, IL-1␤, IL-6 and prostaglandin PGE2 are not stored intracellularly within resting monocytes and macrophages, but synthesized and secreted rapidly and transiently following an inflammatory insult (Janeway and Travers 1997). Transcriptional control is the key step in regulation of synthesis of cytokines and many other inflammatory factors (Hopkins and Failla 1999, Montgomery and Dallman 1997). Genes for the proinflammatory cytokines TNF-␣, IL-1␤ and IL-6, as well as the gene for inducible cyclooxygenase 2 (PGE2 synthase), contain both nuclear factor (NF)-␬B and NF-IL-6 consensus sequences in their promoter regions (Sorli et al. 1998, Sweet and Hume 1996). Therefore, the suppressed secretion of the inflammatory mediators may be associated with diminished activities of the transcriptional factors NF-␬B and NF-IL-6. The activities of NF-␬B and NF-IL-6 have been shown to be affected by cellular redox status (Sen and Packer 1996, Sorli et al. 1998). Several observations support the possibility that Cu deficiency alters cell redox status, thereby affecting transcriptional events. Allen et al. (1988) have shown that glutathione synthesis and the ratio of glutathione to oxidized glutathione are increased in Cu-deficient liver and kidney of rats. The enhanced transcription of the fatty acid synthase gene in liver of Cu-deficient rats was offset by restoration of normal thiol redox status in the Cu-deficient liver (Wilson et al. 1997). Hopkins and Failla (1997b) also reported decreased NF-␬B DNA-binding activity in nuclear extracts from liver and spleen of LPS-treated Cu-deficient rats compared with similar extracts prepared from LPS-treated Cu-adequate rats. In summary, Cu deficiency induced in differentiating U937 cells directly suppressed respiratory burst, bactericidal activity and secretion of inflammatory mediators TNF-␣, IL-1␤, IL-6 and PGE2 without marked alterations in general cell characteristics, metabolic state and extent of maturation. Treatment of U937 cells with a low dose of tet provides a useful model for examining further the specific biochemical roles of Cu in effector activities of monocytes and macrophages. Ongoing studies are focused on defining the biochemical basis for effects of Cu deficiency on the differentiation and activation of monocytes and macrophages using the U937 cell model.

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