Augmented metalloproteinase activity and acute lung injury in copper-deficient rats

Alex B. Lentsch1, Atsushi Kato1, Jack T. Saari2, and Dale A. Schuschke3

Departments of 1 Surgery and 3 Physiology and Biophysics, University of Louisville School of Medicine, Louisville, Kentucky 40292; and 2 Grand Forks Human Nutrition Research Center, Agricultural Research Service, United States Department of Agriculture, Grand Forks, North Dakota 58202


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Dietary copper is required for normal function of >30 mammalian enzyme systems. Copper deficiency causes a number of cardiovascular defects as well as impaired immune cell function. Little is known regarding the effects of copper deficiency on acute inflammatory responses, but this topic is relevant because many members of the Western population receive less than the recommended dietary allowance of copper. In the current studies, we investigated the effects of dietary copper deficiency on acute lung injury induced by intrapulmonary deposition of IgG immune complexes. Weanling male Long-Evans rats were fed diets either adequate (5.6 µg/g) or deficient (0.3 µg/g) in copper. IgG immune complex lung injury was greatly increased in copper-deficient rats as determined by lung vascular leakage of albumin and histopathology. However, no change was observed in either the lung content of tumor necrosis factor-alpha or lung neutrophil accumulation. Lungs from copper-deficient rats had much higher levels of matrix metalloproteinase (MMP)-2 and MMP-9 than did copper-adequate control animals. This increased activity was not attributable to alveolar macrophages or neutrophils. These data suggest that the augmented lung injury caused by copper deficiency is due to increased pulmonary MMP-2 and MMP-9 activity and not a generalized amplification of the inflammatory response.

inflammation; gelatinase; neutrophils; cytokines


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

COPPER IS A MICRONUTRIENT that is essential for the normal function of a large number of mammalian enzyme systems. Experimental animal models of copper deficiency have demonstrated impaired enzyme activity in association with severe defects in cardiovascular function including altered vascular tone, cardiac hypertrophy, hypotension, and hypercholesterolemia (24). Recent evidence (1, 10) also suggests that adequate intake of copper is necessary for normal immune function. Even marginal copper deficiency, which does not result in cardiovascular defects, causes markedly impaired function of neutrophils and lymphocytes. These studies showed that decreased copper intake reduced lymphocyte proliferation by decreasing the production of interleukin-2 and reduced the ability of neutrophils to generate superoxide anion and kill ingested microorganisms. A subsequent study (14) has shown that copper is essential for the normal maturation of neutrophils.

The current recommended dietary allowance for copper is 900 µg/day, although numerous dietary surveys indicate that many typical Western diets provide less than this amount (16). Although no overt symptoms of copper deficiency have been identified in the general population, compromised immune function in individuals with reduced copper status may increase their susceptibility to infection or inflammatory injury. In neonatal rats, reduced copper intake has been associated with a predisposition to development of acute respiratory distress syndrome (ARDS) (25). However, given the adverse effects of copper deficiency on neutrophil development and function (1, 10, 14) and the known participation of neutrophils in the pathogenesis of ARDS and similar syndromes (29, 33), it might be expected that reduced copper intake would result in a diminished lung inflammatory response.

In the current study, we examined the effects of copper deficiency on the lung inflammatory response to intrapulmonary deposition of IgG immune complexes. This model has been well characterized in rats and bears many similarities to acute lung injury induced by infection or trauma (11, 20, 34). Central to the development of lung inflammation in this model is the pulmonary production of tumor necrosis factor-alpha (TNF-alpha ) (32). This cytokine appears to stimulate other inflammatory pathways that facilitate the recruitment of neutrophils from the vascular compartment into the lung parenchyma and airspaces (29). The ensuing lung injury, characterized by increased vascular permeability and alveolar hemorrhage, is thought to be mediated by oxidants and proteases released by neutrophils, lung macrophages, and activated lung parenchymal cells. Our results demonstrate that copper deficiency results in greatly augmented lung injury independent of TNF-alpha production or neutrophil recruitment. We found markedly increased activity of matrix metalloproteinase (MMP)-2 and MMP-9 in lungs from copper-deficient rats that could not be attributed to alveolar macrophages or neutrophils. Based on these data, it appears that increased lung injury in copper-deficient rats is caused by augmented MMP activity within the lung compartment.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals and diet. Male weanling Long-Evans rats were purchased from Charles River Laboratories (Wilmington, MA). On arrival, rats were housed individually in stainless steel cages in a temperature- and humidity-controlled room with a 12:12-h light-dark cycle. The rats were given free access to distilled water and to one of two purified diets for 4 wk. The basal diet (13) was a casein-sucrose-cornstarch-based diet (TD 84469, Teklad test diets, Madison, WI) containing all known essential vitamins and minerals except for copper and iron. The copper-adequate (CuA) diet consisted of the basal diet (940 g/kg total diet) along with safflower oil (50 g/kg) and a copper-iron mineral mix that provided 0.22 g ferric citrate (16% Fe) and 24 mg cupric sulfate (monohydrate)/kg diet. The copper-deficient (CuD) diet was the same except for the replacement of copper with cornstarch in the mineral mix. Diet analysis by atomic absorption spectrophotometry indicated that the CuA diet contained 5.56 mg copper/kg diet, and the CuD diet contained 0.33 mg copper/kg diet. Parallel assays of National Institute of Standards and Technology (NIST, Gaithersburg, MD) reference samples (citrus leaves, no. 1572) yielded values within the specified range, which validated our copper assays.

Hepatic copper content. The median lobe of the liver was removed, weighed, and frozen at -20°C for subsequent copper analysis. Liver samples were lyophilized and digested in nitric acid and hydrogen peroxide (H2O2) as previously described (21). Hepatic copper concentrations were assessed by inductively coupled argon plasma emission spectrometry (Jarrell-Ash model 1140, Waltham, MA). Parallel assays of reference samples from the NIST (bovine liver, no. 1477a) yielded mineral contents within the specified range.

Lung Cu/Zn superoxide dismutase activity. Analysis of lung homogenates for Cu/Zn superoxide dismutase (SOD) activity was initiated by treatment with 0.4 volume of a solution of ethanol and chloroform (25:15) to precipitate Mn SOD (22). This solution was mixed well and centrifuged at 5,000 g for 15 min. Aliquots of clear supernatant were dialyzed against deionized water (4°C, 12,000 md wt exclusion membrane). Cu/Zn SOD activity of the dialysate was measured spectrophotometrically (Beckman Coulter model DU 650, Fullerton, CA) with a kit (OxyScan SOD-525) from OXIS International (Portland, OR). The method is based on the ability of SOD to accelerate autoxidation of a proprietary reagent to a chromophore, with maximum absorbance at 525 nm.

IgG immune complex-induced lung injury. Rats were anesthetized with ketamine hydrochloride (150 mg/kg ip). For measurement of pulmonary vascular permeability, rats received an intratracheal administration of PBS, pH 7.4, or 1.5 mg of rabbit polyclonal IgG anti-BSA (ICN Biomedical, Costa Mesa, CA) in a volume of 0.3 ml of PBS. Immediately thereafter, 10 mg of BSA (<1 ng endotoxin/mg) containing trace amounts of 125I-labeled BSA in 0.5 ml of PBS were injected intravenously. Four hours after IgG immune complex deposition, rats were exsanguinated, the pulmonary circulation was flushed with 10 ml of PBS by pulmonary arterial injection, and the lungs were surgically dissected. The extent of lung injury was quantified by calculating the lung permeability index by dividing the amount of radioactivity (125I-labeled BSA) in the perfused lungs by the amount of radioactivity in 1.0 ml of blood obtained at the time of death.

Lung myeloperoxidase content. Whole lung myeloperoxidase (MPO) activity was quantitated as described previously (30). Briefly, 100 mg of lung tissue were homogenized and diluted in 50 mM potassium phosphate buffer containing 0.5% hexadecyltrimethylammonium bromide, pH 6.0. After sonication and two freeze-thaw cycles, samples were centrifuged at 4,000 g for 30 min. The supernatants were reacted with H2O2 (0.3 mM) in the presence of tetramethylbenzidine (1.6 mM). MPO activity was assessed by measuring the change in absorbance at 655 nm with human MPO used as a standard.

Bronchoalveolar lavage fluid content of TNF-alpha . Bronchoalveolar lavage (BAL) fluid was collected by instilling and withdrawing 5 ml of sterile PBS three times from the lungs via an intratracheal cannula. The TNF-alpha content of the BAL fluid was measured with an enzyme-linked immunosorbent assay (ELISA) purchased from BD PharMingen (San Diego, CA).

Substrate-embedded enzymography. SDS-PAGE gels (7.5%) containing 1 mg/ml of gelatin were prepared. Denatured but nonreduced BAL fluid samples or cell supernatants were electrophoresed into the gels at constant voltage. The gels were then washed twice (20 min/wash) in water containing 2.5% Triton X-100 at room temperature and incubated overnight in activation buffer (10 mM Tris · HCl, pH 7.5, 1.25% Triton X-100, 5 mM CaCl2, and 1 µM ZnCl2) at 37°C. Gels were then stained with Coomassie blue for 3 h and then destained. Regions of negative staining indicated the presence of proteinases with gelatinolytic activity.

Western blot analysis. BAL fluid (35 µl) was separated in a denaturing 10% polyacrylamide gel and transferred to a 0.1-µm-pore nitrocellulose membrane. Nonspecific binding sites were blocked with Tris-buffered saline (TBS; 40 mM Tris, pH 7.6, and 300 mM NaCl) containing 5% nonfat dry milk for 2 h at room temperature. Membranes were then incubated overnight at 4°C in 0.5 µg/ml of polyclonal goat anti-MMP-2 or anti-MMP-9 (Santa Cruz Biotechnology, Santa Cruz, CA) in TBS with 0.1% Tween 20 (TBS-T). After three washes in TBS-T, the membranes were incubated for 2 h in 0.15 µg/ml of horseradish peroxidase-conjugated donkey anti-goat IgG (Santa Cruz Biotechnology) at room temperature. Membranes were washed three times in TBS-T, and immunoreactive proteins were detected by enhanced chemiluminescence.

Isolation and culture of rat alveolar macrophages and peripheral blood neutrophils. Rat alveolar macrophages from otherwise normal rats fed either a CuA or CuD diet were isolated by BAL. Cells were pelleted by centifugation and plated in DMEM (1 × 106 cells/ml) supplemented with 0.2% BSA. After being allowed to adhere for 1 h, the cells were washed with fresh medium to remove nonadherent cells. Peripheral blood neutrophils were isolated by dextran and Percoll separation. The remaining red blood cells were hypotonically lysed, and the neutrophils were washed and resuspended in DMEM (1 × 106 cells/ml) supplemented with 0.2% BSA. After isolation, alveolar macrophages or blood neutrophils were cultured for 2 h in the absence and presence of TNF-alpha (10 ng/ml) or lipopolysaccharide (10 µg/ml). Supernatants were then harvested and subjected to substrate-embedded enzymography.

Statistical analysis. All data are expressed as means ± SE. Data were analyzed with a one-way analysis of variance with subsequent Student-Newman-Keuls test. Differences were considered significant when P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Establishment of copper deficiency. Rats fed a diet deficient in copper for 4 wk had significantly less hepatic copper and were anemic (Table 1). These markers are indicative of copper deficiency. In the Long-Evans rats used for these experiments, the CuD diet slowed weight gain; these rats weighed ~10% less at the time of experimentation than rats fed a CuA diet (Table 1). Furthermore, CuD rats had significantly less Cu/Zn SOD activity in lung tissue than rats fed a CuA diet (Table 1), indicating a potentially altered redox state in the lung.

                              
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Table 1.   Indexes of copper status

Effects of copper deficiency on IgG immune complex-induced lung injury. Because lung SOD activity was reduced in rats fed a CuD diet and because acute inflammatory reactions are often dependent on redox status, we sought to determine whether the lack of dietary copper altered the acute lung injury induced by IgG immune complexes. The extent of lung injury was determined by pulmonary vascular leakage of 125I-albumin. In rats fed the CuA diet, deposition of IgG immune complexes caused a 130% increase in the lung permeability index compared with that of rats receiving intratracheal PBS (Fig. 1). Interestingly, in rats fed the CuD diet, IgG immune complex deposition resulted in a 354% increase in the permeability index compared with similarly fed rats receiving intratracheal PBS. This augmented lung injury was evident by a more than twofold increase in the permeability index of CuD rats versus CuA rats. There was no difference in the lung permeability index in the PBS control rats fed either diet.


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Fig. 1.   Effects of copper deficiency on IgG immune complex-induced lung vascular permeability. The permeability index was calculated 4 h after intratracheal administration of PBS or IgG immune complexes (IgG-IC) as described in MATERIALS AND METHODS. Values are means ± SE; n = 5 rats/group. Significantly different (P < 0.01) from: *PBS control rats; dagger PBS control and copper-adequate (CuA) rats receiving IgG-IC.

Lung injury was also assessed by histological analysis. Rats that were fed either the CuA or CuD diet and receiving intratracheal PBS had normal lung architecture (Fig. 2, A and B). Lungs from CuA rats undergoing IgG immune complex injury displayed increased interstitial cellularity, disruption of alveolar integrity, and intra-alveolar accumulation of erythrocytes and neutrophils (Fig. 2C). Lungs from CuD rats that underwent IgG immune complex injury exhibited a much greater extent of alveolar hemorrhage than did their CuA counterparts (Fig. 3D). In addition, IgG immune complex-injured lungs from CuD rats consistently showed evidence of copious quantities of proteinaceous fluid within the alveoli. Lung sections from both CuA and CuD rats showed similar amounts of neutrophils.


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Fig. 2.   Effects of copper deficiency on lung histopathology. Lung sections were obtained from rats 4 h after intratracheal administration of PBS or IgG-IC. Normal lung architecture was observed in rats receiving PBS and fed either a CuA (A) or copper-deficient (CuD; B) diet. C: in lungs from CuA rats, IgG-IC deposition caused marked neutrophil accumulation and intra-alveolar hemorrhage. D: in lungs from CuD rats, IgG-IC deposition caused much more intra-alveolar hemorrhage, disruption of alveolar integrity, and the presence of large amounts of proteinaceous fluid within alveoli. Hematoxylin and eosin staining; original magnification, ×20.



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Fig. 3.   Effects of copper deficiency on bronchoalveolar lavage (BAL) fluid content of tumor necrosis factor (TNF)-alpha . BAL fluid was harvested 4 h after intratracheal administration of PBS or IgG-IC as described in MATERIALS AND METHODS. Values are means ± SE; n = 4 rats/group. *P < 0.01 compared with PBS control animals.

Effects of copper deficiency on lung production of TNF-alpha and lung neutrophil accumulation. The development of the lung inflammatory response induced by IgG immune complexes requires the pulmonary production of the cytokine TNF-alpha , and the ensuing lung injury is heavily dependent on the accumulation of neutrophils in the lung vasculature and airspaces (32). To determine how copper deficiency augments the lung inflammatory response, we examined the pulmonary production of TNF-alpha and the lung accumulation of neutrophils in both CuA and CuD rats. BAL fluid levels of TNF-alpha were near the lower limit of detection of the ELISA in rats receiving intratracheal PBS (Fig. 3). IgG immune complex deposition caused significant increases in BAL fluid levels of TNF-alpha in both CuA and CuD rats. However, there was no significant difference between these groups.

The extent of neutrophil accumulation was assessed by MPO activity in the lung. Surprisingly, rats fed the CuD diet and receiving intratracheal PBS had significantly greater levels of MPO activity than their CuA counterparts (Fig. 4). Intrapulmonary deposition of IgG immune complexes resulted in large increases of lung MPO activity in both CuA and CuD rats. Similar to the findings in BAL fluid TNF-alpha levels, there was no significant difference between these groups.


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Fig. 4.   Effects of copper deficiency on IgG-IC-induced lung neutrophil accumulation. Lung myeloperoxidase (MPO) content was determined 4 h after intratracheal administration of PBS or IgG-IC as described in MATERIALS AND METHODS. Values are means ± SE; n = 5 rats/group. Significantly different (P < 0.01) from: *CuA rats receiving PBS; dagger PBS control rats.

Effects of copper deficiency on MMP activity and expression. MMPs are involved in the parenchymal damage that occurs during lung inflammatory injury (3, 8, 9). Others have shown that changes in the cellular redox state or in expression of Cu/Zn SOD can dramatically affect the expression and/or activation of MMPs (4, 5, 19). Because we observed that CuD rats had significantly less Cu/Zn SOD activity than rats fed a CuA diet (Table 1), we analyzed BAL fluid for the presence of MMPs with gelatinolytic activity. In CuA rats receiving intratracheal PBS, no MMP activity was observed (Fig. 5). In contrast, in BAL fluid from CuD rats, a 72-kDa band was seen, suggesting that copper deficiency may increase the release of MMP-2 (72-kDa gelatinase A) in the lung in unstimulated rats. Intrapulmonary deposition of IgG immune complexes resulted in large amounts of both MMP-2 and a 92-kDa band that likely represents MMP-9 (92-kDa gelatinase B). Interestingly, there was much more MMP-2 and MMP-9 activity present in the BAL fluid from CuD rats.


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Fig. 5.   Gelatin zymography of BAL fluid harvested 4 h after intratracheal administration of PBS or IgG-IC in CuA and CuD rats. Results represent duplicate experiments.

To determine whether the increased MMP activity observed in CuD rats was due to increased enzyme activity or increased expression of enzyme, we analyzed BAL fluid by Western blot for MMP-2 and MMP-9. In BAL fluid from both CuD and CuA rats that received intratracheal PBS, neither MMP-2 or MMP-9 was detectable (data not shown). However, immunoreactive proteins corresponding to 72-kDa MMP-2 and 92-kDa MMP-9 were detected in BAL fluid from rats that received intrapulmonary deposition of IgG immune complexes (Fig. 6). For both MMP-2 and MMP-9, there was markedly more protein in BAL fluid from CuD rats, suggesting that copper deficiency increases the expression of these enzymes.


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Fig. 6.   Protein expression of matrix metalloproteinase (MMP)-2 and MMP-9 in BAL fluid harvested 4 h after intratracheal administration of IgG-IC in CuA and CuD rats. Western blot analysis was conducted as described in MATERIALS AND METHODS.

MMP-2 and MMP-9 are released by a variety of relevant cell sources, including alveolar macrophages, lung epithelial cells, and neutrophils (6, 8, 15). To determine whether cells from CuD rats released more MMP-2 and MMP-9 than CuA rats, alveolar macrophages and peripheral blood neutrophils were harvested from otherwise unmanipulated rats. After 2 h of culture, supernatants were harvested and processed for gelatin zymography. As shown in Fig. 7, similar amounts of MMP-2 and MMP-9 were found in supernatants from alveolar macrophages and neutrophils obtained from rats fed CuA and CuD diets. Similarly, no differences were detected between dietary groups in supernatants from cells stimulated for 2 h with TNF-alpha or lipopolysaccharide (data not shown).


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Fig. 7.   Gelatin zymography of culture supernatants from alveolar macrophages harvested by BAL (top) and peripheral blood neutrophils (bottom) from CuA and CuD rats. Cell supernatants were harvested after 2 h of culture. Results represent duplicate experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IgG immune complex-induced lung inflammation is a well-characterized model of acute lung injury in which the deposition of IgG immune complexes in small airways and alveoli causes activation of lung macrophages. Activated alveolar macrophages produce and secrete proinflammatory cytokines, including TNF-alpha (32). A primary function of TNF-alpha is to promote the inflammatory response by stimulating other lung cells to produce neutrophil-attracting C-X-C chemokines and cause upregulation of cellular adhesion molecules on the pulmonary vascular endothelium (29). Through a series of complex coordinated interactions, chemokines and adhesion molecules facilitate the adhesion and transmigration of neutrophils from the vascular lumen into the lung interstitium and airspaces. The products of these activated neutrophils and lung macrophages, which include oxidants and proteases, result in damage to lung cells and matrix components. In the current studies, we used this well-defined model of lung injury to investigate the effects of copper deficiency on the induction and propagation of the lung inflammatory response.

IgG immune complex lung injury was much more severe in CuD rats as determined by pulmonary vascular leakage and histopathological analysis. Interestingly, there was no difference in the extent of neutrophil accumulation in the injured lungs from rats fed either a CuA or CuD diet. These data suggest that the enhanced lung injury observed in CuD rats was independent of neutrophils. Consistent with these findings, when intrapulmonary levels of TNF-alpha protein were measured, there were no differences between CuA and CuD rats. Thus it appears that copper deficiency does not amplify the inflammatory response by increasing TNF-alpha production or subsequent events leading to the recruitment of neutrophils.

Copper deficiency did result in reduced activity of Cu/Zn SOD in lung. A previous study (12), however, suggested that there is no direct protective role for SOD in IgG immune complex lung injury. Exogenous administration of SOD had virtually no effect on lung permeability or histopathology in this model. Despite these findings, depressed SOD activity may result in a shift toward a prooxidant environment in the lung. The depression of Cu/Zn SOD activity is representative of a general shift to a prooxidant environment in CuD animals that includes depression of other antioxidant enzymes as well as a change in the metabolism of iron that promotes free radical formation (23). A prooxidant milieu may augment the generation of other factors that contribute to lung injury, such as MMPs. Our current data demonstrate that CuD rats, which have significantly less lung SOD activity, have augmented production or activity of MMP-2 and MMP-9. Both of these enzymes are known to be positively regulated by reactive oxygen species such as H2O2 and superoxide anion (4, 5, 19). Conversely, both MMP-2 and MMP-9 are inhibited by tissue inhibitors of metalloproteinases (TIMPs). However, we found no difference in the mRNA expression of TIMP-1 or TIMP-2 in lungs from rats fed a CuA or CuD diet (unpublished observations).

In the context of acute lung injury, a number of cells may produce MMP-2 and MMP-9, including lung epithelial and endothelial cells, alveolar macrophages, and recruited neutrophils (4, 6, 8, 15). Copper deficiency is known to impair the function of neutrophils, macrophages, and endothelial cells. Neutrophils from CuD rats show diminished respiratory burst and bactericidal activity (1, 10). Similar effects are seen in macrophages from CuD rats (2). Endothelial cell dysfunction induced by copper deficiency appears to be related to suppressed intracellular calcium mobilization (26). In the current studies, no differences in MMP-2 or MMP-9 activity were observed in neutrophils or alveolar macrophages from CuD rats. These findings exclude the possibility that these cells are responsible for the increased MMP-2 and MMP-9 activity found in the BAL fluid of rats fed a CuD diet. Furthermore, by Western blot analysis of BAL fluid, we demonstrated a marked increase in the protein content of MMP-2 and MMP-9. Because this model does not involve lung recruitment of other leukocytes, it is likely that copper deficiency somehow alters lung epithelial and/or endothelial cells to increase their production of MMP-2 and MMP-9. Although our data strongly suggest that copper deficiency results in augmented production of MMPs, an in vitro study (28) showed that copper may also inhibit the activity of MMPs.

A role for MMPs has been established in various animal models of neutrophil-dependent and neutrophil-independent lung injury (3, 8, 9). Clinically, enhanced activity of MMP-2 and MMP-9 is a common observation in patients with ARDS and chronic obstructive pulmonary disease, and it is thought that the enhanced activity of MMP-2 and MMP-9 contributes significantly to the pathogenesis of these syndromes (6, 27, 31). Based on our current data, it is tempting to suggest that individuals who are marginally copper deficient may be predisposed to an exaggerated lung inflammatory response after an insult leading to acute lung injury (i.e., trauma, infection). This concept may also extend to premature infants. It is well documented that premature infants have lower serum copper concentrations than their full-term counterparts (18). Furthermore, premature infants continue to have low serum copper concentrations for up to 6 mo after birth, whereas those of full-term infants quickly reach adult levels (17). Respiratory distress is a major cause of morbidity among premature infants (7), and thus it is inviting to suggest an association between copper status and the risk of pulmonary dysfunction in premature infants.

The current study demonstrates that copper deficiency dramatically enhances lung injury after intrapulmonary deposition of IgG immune complexes. This enhanced injury is not due to a generalized amplification of the inflammatory response because production of the proinflammatory cytokine TNF-alpha and the lung recruitment of neutrophils were unchanged. The mechanism by which copper deficiency increases lung injury appears to be augmented MMP-2 and MMP-9 activity in the lung compartment. Likely target cells are lung epithelial and endothelial cells. A more detailed understanding of the effects of dietary copper intake on the acute inflammatory response may yield clues to the factors contributing to pulmonary syndromes such as ARDS.


    ACKNOWLEDGEMENTS

We thank Sharon Young and Gwen Dahlen for expert technical assistance.


    FOOTNOTES

This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-55030-02.

The US Department of Agriculture, Agricultural Research Service, Northern Plains Area, is an equal opportunity/affirmative action employer, and all agency services are available without discrimination.

Address for reprint requests and other correspondence: D. A. Schuschke, Dept. of Physiology and Biophysics, HSC-A1103, Louisville, KY 40292 (E-mail: daschu01{at}gwise.louisville.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 6 November 2000; accepted in final form 12 March 2001.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Lung Cell Mol Physiol 281(2):L387-L393