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The Journal of Nutrition Vol. 128 No. 11 November 1998,
pp. 1855-1860
Food Science and Human Nutrition Department, University of Florida, Gainesville, FL 32611
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ABSTRACT |
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Abstract
Introduction
Methods
Results
Discussion
References
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The objective of this study was to examine the role of copper in neutrophil development and function. Mice were made copper deficient by feeding dams a diet containing 1.05 µg copper starting at parturition. Control mice were fed the same diet containing 6 µg copper. The pups were weaned to the diet and killed when they were 5-6 wk old. Peripheral blood cell counts, margination and cell maturity were measured. The response to an intraperitoneal injection of lipopolysaccharide (LPS) was also determined. Copper deficiency resulted in twice as many neutrophils and fewer than half the number of lymphocytes. Half as many cells in copper-deficient mice expressed Ly-6G, a granulocytic marker of cell maturity. In addition, copper-deficient cells expressed only half the amount of Ly-6G per cell than was expressed by copper-adequate cells. This suggested that the cells were younger, or arrested in their maturation as a result of copper deficiency. An arrest of maturation has been proposed as the cause of neutropenia in human copper deficiency. Injection of LPS in copper-adequate mice resulted in twice as many Ly-6G-expressing cells in the periphery. LPS injection into copper-deficient mice resulted in a severe leukopenia but did not influence Ly-6G expression any more than did copper deficiency alone. LPS treatment caused an increase in myeloperoxidase activity associated with the lungs of copper-deficient mice. The results suggest that although the neutrophils of copper-deficient mice are immature, they can be sequestered by the lung when stimulated to do so.
KEY WORDS: copper deficiency · mice · neutrophil · differentiation · immunity
A lack of copper results in neutropenia in humans receiving parenteral nutrition lacking copper. Individuals described in these case studies received long-term parenteral nutrition for months to years before being diagnosed as copper deficient. Neutropenia was reversed upon reintroduction of copper (Percival 1995 Neutrophils, apart from acting as circulating phagocytes, play an important role in the inflammatory response. After injury, the bone marrow releases more neutrophils into the circulation. A few of these newly released neutrophils are immature banded cells. Neutrophils migrate to the site of damage, penetrate the tissues and kill invading microorganisms. How copper deficiency affects neutrophil responses to inflammatory stimuli is not well understood and is also a part of this research.
To determine the relationship between dietary copper intake and peripheral blood neutrophils, a murine model was studied. The objectives were to characterize the peripheral blood neutrophil response to copper deficiency and, subsequently, to determine the response of neutrophils to an inflammatory stimulus.
Mice.
Procedures were approved by the Institutional Animal Care and Use Committee of the University of Florida. Timed pregnant outbred ICR strain mice were purchased from Harlan Sprague Dawley (Indianapolis, IN) at 8 wk of age; upon arrival, mice were acclimated to a standard nonpurified diet (Harlan Teklad 4% mouse/rat diet, 7001, Madison, WI) for at least 5 d. The mice had free access to the diet and to distilled, deionized water. On the day of parturition, the dams were fed either a copper-deficient (CuD)5 diet, 1.05 ± 0.13 µg/g or a copper-adequate (CuA) diet, 6.4 ± 0.32 µg/g; diets were prepared by Dyets (Bethlehem, PA) using the AIN 93G formulation (Reeves et al. 1993 Lipopolysaccharide treatment.
Pups were randomly assigned to treatment groups. One half of each group of mice was injected intraperitoneally with lipopolysaccharide (LPS) in isotonic saline 17 h before killing. A preliminary study showed that neutrophilia resulted 17 h after injection and occurred before the peak increase in ceruloplasmin activity or serum copper levels. The mice were injected with 50 µg of LPS from Escherichia coli 026:B6 (cell cultured, tested and chromatographically purified by gel filtration) (Sigma Chemical, St. Louis, MO). Control mice were injected intraperitoneally with saline.
Blood collection.
Blood was collected via the inferior vena cava after opening the body cavity. A 1-mL syringe containing EDTA was used to bleed the mice. Whole blood (60 µL) was set aside for flow cytometry analysis. A 300-µL blood sample was placed in lithium heparin microtainer tubes (Becton Dikenson, San Jose, CA) for complete blood count analyses, including hemoglobin, hematocrit and fibrinogen, by the University of Florida Veterinary Medical Center Hematology Department. The remaining blood was centrifuged at 350 × g for 20 min at room temperature. Plasma was immediately frozen at Flow cytometry analysis.
A direct two-color immunofluorescence staining technique using whole blood was employed to quantitate expression of the cell-surface marker Ly-6G in peripheral blood (McCarthy and Macey 1993 Superoxide dismutase assay.
The activity of Cu/Zn-superoxide dismutase (Cu/Zn-SOD, EC 1.15.11) was measured in erythrocytes and lung tissue as reported previously (Percival 1993 Myeloperoxidase activity in lung.
Lung myeloperoxidase (MPO, EC 1.11.1.7) activity was determined by an adaptation of the method used by Grisham et al. (1990) Ceruloplasmin oxidase activity assay.
Cp (EC 1.16.3.1) activity was determined by the method of Schosinsky et al. (1974) Statistical methods.
All comparisons between groups were carried out using SigmaStat (version 1.01), produced by Jandel Scientific (San Rafael, CA). Significantly different means after one-way ANOVA were detected by a Student-Newman-Keuls multiple comparison test. When unequal variances were found, data were log transformed. When normality failed, a one-way ANOVA was performed on ranks. All data are expressed as means ± SD. The probability level at which differences were considered significant was P < 0.05.
Indices of copper status of the mice are presented in Table 1. All measures were significantly different in the copper-deficient compared with the copper-adequate mice. The mice sustained signs that are commonly found in copper deficiency, such as enlarged heart, lower copper levels, reduced copper-dependent enzymes and anemia.
The mice nursed by dams fed the copper-deficient diet and then weaned to the copper-deficient diet were very copper deficient. The values reported in Table 1 are in good agreement with values reported by Prohaska's group (Lukasewycz and Prohaska, 1990
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
). Studies in these copper-deficient humans suggested that copper is essential for normal granulopoiesis. It has been shown in many species that resistance to infection is impaired in copper deficiency (Prohaska and Failla 1993), and ineffective granulopoiesis could be a potential cause of this impairment. Neutrophils elicited from copper-deficient mice produce less oxidative burst, although their ability to migrate (chemotaxis) and engulf (phagocytosis) microorganisms is not affected (Babu and Failla 1990, Boyne and Arthur 1981). Impaired oxidative burst may also be explained by ineffective granulopoiesis. Nearly all immune cells are affected by copper deficiency. It is our goal to understand the influence of copper on neutrophils and on granulopoiesis.
MATERIALS AND METHODS
Abstract
Introduction
Methods
Results
Discussion
References
). At weaning (3 wk old), genders were separated and the mice were fed the same diets as their dams for an additional 2-3 wk, or until they were 5-6 wk old. Only male mice were studied. Mice were housed in polycarbonate cages in an environmentally controlled room (12-h light:dark cycle, 25°C). Water bottles were fitted with polyethylene stoppers and stainless steel sipper tubes; distilled, deionized water was used throughout the study. Before the mice were killed, they were transferred to wire-bottomed cages and food was withheld overnight. Copper-deficient mice did not exhibit outward signs of infection, and spot checks for systemic bacteria by culturing whole blood onto blood agar plates and into brain-heart infusion broth were negative. When the mice reached 5-6 wk of age, they were anesthetized with an intraperitoneal injection of ketamine (100 mg/kg body weight) and xylazine (10 mg/kg) and blood collected via the inferior vena cava. After exsanguination, the mice were killed by cervical dislocation.
20°C to be used later for ceruloplasmin (Cp) activity and copper analysis.
). Whole blood was used to avoid density gradient separation of the leukocytes. Separation techniques have been shown to increase the expression of some markers on the cell surface. Whole blood in EDTA (15 µL) was mixed with 250 µL of LDS-751 (Exciton, Dayton, OH). The working solution of LDS-751 was prepared by diluting the stock solution (0.2 g/L, wt/v, in methanol) 1:100 in Hanks' balanced salt solution (HBSS). LDS-751 binds to nuclear DNA; therefore it does not label erythrocytes or platelets and they can be gated out during data acquisition. Samples were placed on ice and incubated with 5 µL of antibody conjugated with fluorescein isothiocyanate (FITC) at 4° C. Samples were adjusted to 300 µL with 1% fetal bovine serum in HBSS. Monoclonal antibodies to Ly-6G (isotype Rat IgG2
) and the corresponding IgG2 isotype control were obtained from Pharmingen, San Diego, CA. Sample analysis was conducted within 5 h. Analyses of labeled cell samples were done with a FACScan Flow cytometer with an argon laser (FACScan, Becton-Dickinson, San Jose, CA). Nonnucleated cells were excluded from data acquisition by gating at ~100 units of fluorescence, based on the LDS-751 fluorescence. Ten thousand intact cells that fluoresced above 100 units were acquired for each measurement. Graphic plots and statistics for flow cytometry analyses were produced using WinMDI software, Version 2.5, build #6 (developed by Joe Trotter of The Scripps Research Institute, La Jolla, CA). "Mean fluorescence" reflects the average fluorescence of the subpopulation of cells having fluorescence greater than the background fluorescence of the isotype controls. "Percent gated" refers to the percentage of cells of the total 10,000 nucleated cells acquired during FACS analysis that was in the region of specifically bound Ly-6G antibody.
). Lung tissue preparation was slightly modified (De Haan et al. 1994). After thawing on ice, lung tissue (~0.05 g) was homogenized in 300 µL of Tris buffer [50 mmol/L Tris, 1 mmol/L diethylenetriaminepentaacetic acid (DPTA), pH 8.2]. Samples were then sonicated on ice 10 times, at 1 cycle/s, 70% duty cycle, and then centrifuged at 100,000 × g for 1 h at 4°C (TLA 100.4 fixed angle rotor, Optima Ultra Centrifuge, Beckman, Fullerton, CA). Protein was determined by the Bradford Protein Assay (Bio-Rad Laboratories, Richmond, CA).
. Briefly, the tissue was minced and homogenized at a 10% (wt/v) in 20 mmol/L KH2PO4 with 1 mmol/L EDTA at pH 7.4. Samples were diluted to 1.5 times their initial volume in the same buffer and centrifuged at 10,000 × g at 4°C for 20 min. The pellet was reconstituted to a final volume of 1 mL with 50 mmol/L acetic acid containing 5 g/L hexadecyltrimethyl ammonium hydroxide (HETAH) at pH 6.0. The sample was then rehomogenized, sonicated, frozen and thawed twice on dry ice. The sample was centrifuged at 10,000 × g at 4°C for 20 min. The supernatant was assayed by measuring the H2O2 -dependent oxidation of 3,3',5,5'-tetramethylbenzidine (TMB). This yielded a blue chromogen possessing a wavelength maximum of 655 nm. Samples (100 µL) were added to a reaction buffer containing 315 µL of 0.8 mol/L KH2PO4, pH 5.4, to which 25 µL of 10% HETAH and 50 µL of 16 mmol/L TMB in dimethylformamide were added. The sample was incubated in a shaking water bath at 37°C for 5 min. The reaction was initiated by adding 10 µL of 30 mmol/L H2O2 and incubated for 3 min at 37°C. Catalase (10 µL) was added, followed by the sequential addition of 2 mL of 0.2 mol/L sodium acetate. Units of myeloperoxidase activity were calculated from a standard curve that used peroxidase enzyme EC 1.11.1.7 (P-8375, Sigma Chemical) as the standard enzyme.
using o-dianisidine. Cp oxidase activity was reported in IU, and determined by calculating the molar absorptivity after subtracting the 5-min reading from the 15-min reading. Copper in the plasma was determined by graphite furnace atomic absorption spectrophotometry as previously reported (Bae and Percival 1994).
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
View this table:
Table 1.
Copper status in mice fed a copper-deficient (CuD) or copper-adequate (CuA) diet from birth1
View this table:
Table 2.
Peripheral blood cell differentials in copper-adequate (CuA) and copper-deficient (CuD) mice with and
without lipopolysacchanide (LPS) stimulation1
View larger version (21K):
[in a new window]
Fig 1.
Myeloperoxidase activity in the lung tissue of copper-adequate (CuA) and copper-deficient (CuD) mice stimulated or not stimulated with an injection of lipopolysaccharide. Results are expressed means ± SD, n = 6. Different letters (A, B) indicate significantly different means, P < 0.05.
View this table:
Table 3.
Measures of copper status and inflammation in copper-adequate (CuA) and copper-deficient (CuD) mice with and without lipopolysaccharide (LPS) stimulation1
View larger version (36K):
[in a new window]
Fig 2.
Expression of Ly-6G on the surface of peripheral blood cells in copper-adequate and copper-deficient mice stimulated or not stimulated with an injection of lipopolysaccharide. These are representative histogram plots of Ly-6G antibody staining of peripheral leukocytes from whole-blood preparations. The shaded histogram represents the Ly-6G antibody, whereas the open histogram represents the nonspecific bound isotype control antibody. Values for mean fluorescence number of cells were obtained from specifically bound Ly-6G antibody after excluding 95% of the nonspecifically bound control.
View this table:
Table 4.
Flow cytometric analysis of cell surface Ly-6G molecule
on leukocytes of copper-adequate (CuA) and
copper-deficient (CuD) mice with and without lipopolysaccharide (LPS) stimulation1
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
, Prohaska et al. 1983, Prohaska and Lukasewycz 1990) and with those of Mulhern and Koller (Koller et al. 1987, Mulhern and Koller 1988). These two groups used the perinatal mouse model, whereas this study used an immediately postnatal model.
), similar to the functional impairment observed in copper deficiency (Prohaska and Failla1993). However, the phenotypic changes in leukocyte differentials do not occur in iron deficiency as they do in copper deficiency. Iron deficiency may result in a reduction in neutrophils and lymphocytes, but the proportions of cells remain normal (Kriete et al. 1995 and cited in Fairbanks 1994). Others report no change in leukocyte numbers in iron deficiency (Hrycek et al. 1991). Copper deficiency results in a reversal in the proportion of leukocytes from 16% neutrophils and 75% lymphocytes to 51% neutrophils and 44% lymphocytes (Table 2). Data from Rickard et al. (1975) indicated that iron was not a major requirement for granulopoiesis, whereas our preliminary data in bone marrow (unpublished) indicate an important role for copper. Furthermore, changes in immunity due to copper deficiency are detected whether or not anemia is present. In humans (Dunlap et al. 1974) and Hayton et al. (1995) and in rodents (Babu and Failla 1990, Lukasewycz and Prohaska 1990, Prohaska et al. 1983) changes in immune cell function have been demonstrated when hemoglobin and/or hematocrit was low. On the other hand, similar changes in immunity without an accompanying anemia have been shown in humans (Goyens et al. 1985, Higuchi et al. 1988, Sriram et al. 1986), mice (Mulhern and Koller 1988) and cattle (Arthington et al. 1996). We conclude that those changes in immunity due to copper deficiency occur regardless of the hemoglobin levels and, moreover, iron deficiency does not result in changes in the differential profile of leukocytes as does copper deficiency.
). Species difference may be a reason that the mice and cattle become neutrophilic consuming a copper-deficient diet and humans become neutropenic. Another explanation for the differences could be the duration of the copper deficiency. Our study initiated copper deficiency in mice at birth, and it continued until the mice were young adults. Humans who became neutropenic did so as adults, after several months and, in some cases, years of parenteral nutrition before presenting symptoms required attention. It is not known whether humans were neutrophilic at early stages of copper deficiency or whether the mice will eventually become neutropenic. Finally, as one last alternative explanation, we showed that mice become neutropenic after an injection of LPS. It is possible that the neutropenia of copper-deficient humans may be the result of an inflammation or infection, which was the stimulus that initially brought the individual to the clinician who then diagnosed neutropenia. A review of 13 case studies citing copper-deficient neutropenia in 17 people described 13 individuals with some complication involving infection or inflammation (Botash et al. 1992, Dunlap et al. 1974, Fujita et al. 1989, Goyens et al. 1985, Hayton et al. 1995, Karpel and Peden 1973, Phillip et al. 1990, Prasad et al. 1978, Sriram et al. 1986, Summerfield et al. 1992). No complications were mentioned or apparent in four individuals exhibiting neutropenia (Dunlap et al. 1974, Higuchi et al. 1988, Naveh et al. 1981, Zidar et al. 1977).
showed that granulocytes are the predominate population that stained with this antibody. The function of this marker is not known, but Hestdal et al. (1991) clearly showed the correlation between this marker and the maturity of the myeloid lineage. More Ly-6G is expressed on the cell surface of mature cells. In these studies, copper deficiency resulted in twice as many peripheral blood neutrophils, but the expression of Ly-6G was half that of the copper-adequate cells. Not only were there a lower number of cells expressing Ly-6G, but the expression per cell was also lower. This suggests that the peripheral blood neutrophils from the CuD group were less mature compared with controls. It also concurs with the evidence of banded cells in the periphery and supports the idea that copper deficiency resulted in younger, less differentiated cells in the periphery. We could interpret this as an arrest of maturation, the commonly stated postulate for human neutropenia (Dunlap et al. 1974, Hirase et al. 1992, Zidar et al. 1977).
. CuA mice had greater fibrinogen levels when challenged with LPS, but the CuD mice did not mount a significant increase in fibrinogen in response to LPS.
, Boyne and Arthur, 1981). We estimated the adhesion ability of neutrophils from copper-deficient mice by measuring a neutrophil-specific enzyme in lung homogenates after LPS stimulation. Because of the significant increase in MPO activity in the lungs of the copper-deficient mice after LPS stimulation, it is suggested that adhesion of neutrophils to the endothelium is not impaired. This is somewhat surprising because the cells are less mature and are therefore expected to express fewer cell adhesion molecules. However, the ability of these copper-deficient cells to up-regulate surface expression of the adhesion molecules is unknown. We also do not know the stage of maturity achieved by the copper-deficient neutrophils and therefore cannot predict the expression of adhesion molecules that may or may not be expressed due to the age of the cell.
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FOOTNOTES |
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Manuscript received 6 October 1997. Initial reviews completed 4 December 1997. Revision accepted 17 June 1998.
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ACKNOWLEDGMENTS |
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The authors thank Richard A. Satcher and Kelli Herlinger-Garcia for technical assistance and Neal Benson at the UF-ICBR Flow Cytometry Core Laboratory for assistance and helpful discussions.
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LITERATURE CITED |
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Abstract
Introduction
Methods
Results
Discussion
References
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A Comprehensive Treatise, Vol. 8: Nutrition and Immunology (Klurfield, D. M., ed.), pp. 309-332. Plenum Press, New York, NY.This article has been cited by other articles:
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 S. A. Lowndes, A. Adams, A. Timms, N. Fisher, J. Smythe, S. M. Watt, S. Joel, F. Donate, C. Hayward, S. Reich, et al. Phase I Study of Copper-Binding Agent ATN-224 in Patients with Advanced Solid Tumors Clin. Cancer Res., November 15, 2008; 14(22): 7526 - 7534. [Abstract] [Full Text] [PDF]
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