Expression of antioxidant enzymes in human inflammatory cells

¹ Hospital for Children and Adolescents, University of Helsinki, Helsinki; and ² Department of Internal Medicine, University of Oulu, Oulu, Finland

	ABSTRACT

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Because antioxidant enzymes may have an important role in the oxidant resistance of inflammatory cells, we investigated the mRNA levels and specific activities of manganese and copper-zinc superoxide dismutases (Mn SOD and Cu,Zn SOD), catalase (Cat), and glutathione peroxidase, as well as the concentrations of glutathione (GSH) in human neutrophils, monocytes, monocyte-derived macrophages, and alveolar macrophages. Levels of GSH and glutathione peroxidase activity in monocytes were three times higher than in neutrophils, whereas the mRNA of Cat was 50-fold and its specific activity 4-fold higher in neutrophils. Although Mn SOD mRNA levels were higher in neutrophils, enzyme activities, as well as those of Cu,Zn SOD, were similar in all phagocytic cells. Neutrophils lost their viability, assessed by adenine nucleotide depletion, within 24 h ex vivo and more rapidly if GSH was depleted. However, neutrophils were the most resistant cell type to exogenous H₂O₂. In conclusion, high Cat activity of neutrophils appears to explain their high resistance against exogenous H₂O₂, whereas low GSH content and GSH-related enzymes seem to account for the poor survival of human neutrophils.

superoxide dismutase; catalase; glutathione peroxidase; neutrophils; monocytes; macrophages

	INTRODUCTION

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PHAGOCYTIC CELLS, i.e., neutrophils, monocytes, and macrophages, generate reactive oxygen species (ROS) during the respiratory burst by membrane-bound NADPH oxidase (48) and play an important role in defense against microorganisms and various exogenous compounds (5). ROS may, however, damage not only phagocytes but also adjacent tissues (21). Cells are protected against ROS by antioxidant enzymes, such as Mn and Cu-Zn superoxide dismutase (Mn SOD and Cu,Zn SOD), catalase (Cat), and glutathione peroxidase (GPx). Although many studies have focused on the regulation of ROS production in inflammatory cells (45), very little is known about the expression of antioxidant enzymes and/or their role in activated human neutrophils and mononuclear cells.

Antioxidant enzymes have been shown to be modulated during the differentiation of monocytes, but no studies have compared the expression of antioxidant enzymes between various inflammatory cells. Differentiation of human monocytes into macrophages in vitro is accompanied by an increase of Mn SOD and a decrease of Cat and GPx activities (3, 39). Cu,Zn SOD mRNA appears to decrease (4) and GPx gene expression and activity to increase in the human leukemia cell line HL-60 during differentiation into granulocytes or macrophages (49), whereas Mn SOD mRNA remains unchanged (31) or the activity decreases (51). Antioxidant enzymes, most importantly Mn SOD, are also known to be regulated by inflammatory cytokines, oxygen tension, and cigarette smoke in human monocytes, neutrophils, and alveolar macrophages (16, 33, 37, 41, 50, 54); these findings are in line with the studies on the regulation of Mn SOD in numerous other cells (30, 55).

The life span of neutrophils is short compared with that of mononuclear cells. Neutrophils die through apoptosis, in which free radicals play a role (17, 40). It can therefore be hypothesized that the level of intracellular antioxidant enzymes may contribute to the observed differences in the survival of neutrophils and mononuclear cells. In this study, we compared the mRNA levels and/or activities of Mn SOD, Cu,Zn SOD, Cat, GPx, and glutathione reductase (GR) and determined the concentrations of glutathione (GSH) in human monocytes, monocyte-derived macrophages, and neutrophils. Additional experiments were conducted to investigate the importance of these antioxidant enzymes in protection against oxidants generated during the respiratory burst or after exposure to exogenous H₂O₂ in cells in which the antioxidant enzymes had been selectively inhibited.

	MATERIALS AND METHODS

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Cell Isolation and Culture

Monocytes were isolated from buffy coats (Finnish Red Cross, Helsinki, Finland) by a modified method described by Böyum (11). Briefly, buffy coats were obtained 1-2 h after venipuncture. After 1:3 dilution with PBS (Orion, Espoo, Finland), the cell suspension was layered over Ficoll-Paque (Pharmacia, Biotech, Uppsala, Sweden) and centrifuged at 900 g for 30 min. Mononuclear cells were collected at the interface, washed twice with PBS, and suspended in standard RPMI 1640 medium (GIBCO, Paisley, UK) supplemented with L-glutamine (GIBCO), penicillin-streptomycin (GIBCO), and 20% human serum (Finnish Red Cross). Viability tested by trypan blue exclusion was >90%. Mononuclear cells were incubated in collagen-coated (Vitrogen, Collagen, Palo Alto, CA) petri dishes (Falcon, Becton-Dickinson, Plymouth, UK) for 1 h at 37°C. After incubation, nonadherent cells were removed by washing with PBS. Adherent cells were cultured under 5% CO₂ at 37°C for up to 5 days in standard medium, which has been shown to induce maturation of monocytes into so-called monocyte-derived macrophages, the term used in this study. The cells were used for experiments after 0, 24, 72, and 120 h of culture.

Neutrophils were isolated from the heparinized venous blood of healthy volunteers, as previously described (11). Briefly, after sedimentation with 3% dextran (Pharmacia), Ficoll-Paque centrifugation at 400 g for 40 min, and hypotonic lysis of red blood cells, neutrophils were suspended in standard medium and pelleted or used for experiments immediately. To test viability, the cells were cultured in petri dishes under 5% CO₂ at 37°C for up to 24-72 h. Viability after isolation was >90%.

Alveolar macrophages were isolated from bronchoalveolar lavage performed on patients with minor respiratory symptoms (8). Bronchoalveolar lavage fluid was centrifuged at 330 g, and the cells were washed twice with RPMI 1640 medium. Cytocentrifuge preparations indicated that >90% of the cells were macrophages. For further enrichment, cells were layered in petri dishes in RPMI 1640 medium containing 10% fetal bovine serum (GIBCO) and incubated under 5% CO₂ at 37°C for 1 h. Nonadherent cells were removed by washing, and adherent cells were collected in 4 M thiocyanate buffer and frozen at 80°C until analysis.

Northern Blot Analysis

Cells were collected in 4 M thiocyanate buffer and frozen immediately at 80°C. Total RNA was extracted (13), electrophoresed (10 µg/lane), transferred onto Hybond-N nylon filters (Amersham International, Amersham, UK), and cross-linked to the filters by ultraviolet illumination (UV Stratalinker 1800, Stratagene, La Jolla, CA). Filters were hybridized with ³²P-labeled cRNA probes representing nt 596-987 of human Mn SOD (24), nt 127-457 of human Cu,Zn SOD (23), nt 537-2218 of human Cat (44), and nt 533-624 of rat GPx (25), all cloned into the pSP65 vector (Promega, Southampton, UK). Autoradiography was performed at 80°C with Kodak BioMax MR film (Eastman Kodak, Rochester, NY). After autoradiography, the filters were rehybridized with a -actin control probe transcribed from pTRI--actin plasmid (Ambion, Austin, TX). Antioxidant enzyme mRNA expression was quantified relative to actin expression by use of a transmission densitometer (model 331, X-Rite, Grandville, MI). The human Mn SOD, Cu,Zn SOD, Cat, and rat GPx cDNAs were kindly provided by Dr. Y.-S. Ho (Wayne State University, Detroit, MI).

Western Blot Analysis

The cells were mixed with the electrophoresis sample buffer and boiled for 5 min at 95°C. Fifty micrograms of cell protein were applied per lane to a 12% SDS-polyacrylamide gel (34). The gel was electrophoresed for 1.5 h (90 V) at room temperature, and the protein was transferred (45 min, 100 V) onto Hybond enhanced chemiluminescence nitrocellulose membranes (Amersham, Arlington Heights, IL) in a Mini-PROTEAN II cell (Bio-Rad, Hercules, CA). The blotted membrane was incubated with rabbit antibody to recombinant human Mn SOD (1:10,000; a gift from J. D. Crapo, National Jewish Medical and Research Center, Denver, CO) and then with donkey anti-rabbit secondary antibody (1:30,000) conjugated to horseradish peroxidase (Amersham). Mn SOD was detected by an enhanced chemiluminescence system (Amersham), and the luminol excitation was imaged on X-ray film (Eastman Kodak). Cell protein was determined by Bio-Rad DC assay.

Enzyme Activities

Monocytes and monocyte-derived macrophages were detached with trypsin-EDTA (GIBCO). Detached mononuclear cells and neutrophils were pelleted and immediately frozen at 80°C until analysis. Total SOD activity was measured spectrophotometrically by the method of McCord and Fridovich (36). Mn SOD activity was distinguished from Cu,Zn SOD by its resistance to 1 mM potassium cyanide. Cat activity was determined with an oxygen electrode, as described earlier (28). GR activity was analyzed by measuring the oxidation of NADPH in the presence of oxidized GSH (10) and GPx activity by measuring NADPH oxidation in the presence of t-butylhydroperoxide, GSH, and GR (10). Enzyme activities are expressed as units per milligram of protein. Protein was determined by Bio-Rad DC assay.

GSH Content

Cells were collected in 2 N perchloric acid containing 2 mM EDTA. After neutralization with a solution containing 2 M KOH and 0.3 M MOPS, total cellular GSH content was determined spectrophotometrically by measuring the reduction of 5,5'-dithiobis(2-nitrobenzoic acid) (Sigma Chemical) by NADPH in the presence of GR (Sigma Chemical) (2). Cellular GSH content is expressed as nanomoles per milligram of protein.

Pretreatments and Oxidant Exposures

The cells were washed and exposed for 2 h to 10⁷ M formyl-methionyl-leucyl-phenylalanine (FMLP; Sigma Chemical) to induce respiratory burst by activating cell membrane NADPH oxidase (48) or for 4 h to 100-500 µM H₂O₂ (5% CO₂, 37°C). To assess the high-energy nucleotide levels, the cells were first preincubated with 0.1 mM [¹⁴C]adenine (specific activity 284-287 mCi/mmol) in RPMI 1640 medium for 6 h (5% CO₂, 37°C) and then exposed to FMLP or H₂O₂.

To assess the role of different antioxidant enzymes in these cells, additional experiments were conducted in which the cells were pretreated with 10 µM 1-chloro-2,4-dinitrobenzene (Merck, Darmstadt, Germany) for 40 min to conjugate GSH (12) with 100-500 µM buthionine sulfoximine (BSO; Sigma Chemical) for 6 or 16 h to inhibit -glutamylcysteine synthase and deplete GSH (22) or with 100 µg/ml 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) for 15 min to inhibit GR (6) under 5% CO₂ at 37°C. The inhibitors selected for this study have been widely used and also tested in our laboratory (29, 43). GSH was decreased by 96, 94, and 94% with 1-chloro-2,4-dinitrobenzene in neutrophils, monocytes, and monocyte-derived macrophages, respectively (n = 3-5, duplicates).

Assessment of Cell Injury

Trypan blue exclusion. Cell suspension was incubated with trypan blue for 5 min at room temperature. The cells were counted using a Bürker cell counting chamber, and trypan blue-negative cells were considered viable cells.

Adenine nucleotide depletion. High-energy nucleotide levels were assessed in [¹⁴C]adenine-preincubated cells. Prelabeled cells were washed and exposed to FMLP or H₂O₂ in serum-free RPMI 1640 medium (GIBCO), as described previously. After the exposures the medium and the cells extracted with 0.42 N perchloric acid were frozen at 20°C until analysis. Purine nucleotides (ATP, ADP, and AMP) in neutralized cell extracts and nucleotide catabolic products (hypoxanthine, xanthine, and uric acid) in the medium were separated by TLC. The results are expressed as percent distribution of radioactivity (counts/min) between the nucleotides and catabolic products, as described previously (1).

Statistical Analysis

Values are means ± SD. Two groups were compared using the nonparametric Mann-Whitney U test (SPSS for Windows, SPSS, Chicago, IL). P < 0.05 was considered significant.

	RESULTS

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Mn SOD and Cu,Zn SOD

Neutrophils had markedly higher levels of the 1- and 4-kb transcripts of Mn SOD mRNA than mononuclear cells (P < 0.05; Fig. 1A). Large variation was observed in the cells isolated from various individuals, even though the experimental conditions were the same. Mn SOD mRNA, especially its 4-kb transcript, was higher in monocyte-derived macrophages than in freshly isolated monocytes (P < 0.05, n = 3; Fig. 1A). However, the immunoreactive Mn SOD protein by Western blot analysis was highest in monocyte-derived macrophages and similar in monocytes and neutrophils (Fig. 1B). In agreement with the Western blotting analyses, the specific activity of Mn SOD was similar in monocytes and neutrophils and increased in monocytes during the first 3 days in culture (Fig. 1C). The mRNA levels (data not shown) and specific activities of Cu,Zn SOD were similar in all cells investigated (Fig. 1C).

View larger version (25K): [in this window] [in a new window]   Fig. 1.   A: Mn superoxide dismutase (SOD) mRNA levels in freshly isolated neutrophils (Neutro) and monocytes (Mono 0 h), monocytes cultured for 24 h (Mono 24 h), monocyte-derived macrophages (Mono 72 h), and alveolar macrophages (AM). Results, calculated relative to -actin mRNA, are means ± SD of 3 experiments. Hybridization with a human Mn SOD cRNA probe shows evidence of 2 transcripts: 1.0 kb (open bars) and 4.0 kb (filled bars). * P < 0.05 vs. other cells;  P < 0.05 vs. monocytes and alveolar macrophages. B: Western blotting of Mn SOD in neutrophils (N), monocytes (M), monocyte-derived macrophages, and alveolar macrophages. C: activities of Cu,Zn SOD (open bars) and Mn SOD (filled bars) in neutrophils, monocytes, and monocyte-derived macrophages. * P < 0.05 vs. other cells.

H₂O₂-Scavenging Mechanisms and GSH Content

The mRNA of Cat was highest in neutrophils, intermediate in alveolar macrophages, and lowest in monocytes (Fig. 2A). Similarly, the activity of Cat was much higher in neutrophils than in monocytes or monocyte-derived macrophages (Fig. 2B).

View larger version (22K): [in this window] [in a new window]   Fig. 2.   A: catalase mRNA levels in neutrophils, monocytes, monocyte-derived macrophages, and alveolar macrophages. Results, calculated relative to -actin mRNA, are means ± SD of 3 experiments. * P < 0.05 vs. other cells. B: activity of catalase in neutrophils, monocytes, and monocyte-derived macrophages. * P < 0.05 vs. other cells.

The mRNA level and specific activity of GPx were higher in monocytes and monocyte-derived macrophages than in neutrophils (Fig. 3). GR activity was 45.4 ± 7.6 mU/mg protein in monocytes, 38.8 ± 16.8 mU/mg protein in monocyte-derived macrophages, and 30.1 ± 7.4 mU/mg protein in neutrophils, the difference between freshly isolated monocytes and neutrophils being significant (P < 0.05, n = 6-9). Total GSH was significantly higher in monocytes and in monocyte-derived macrophages than in neutrophils (Fig. 4).

View larger version (24K): [in this window] [in a new window]   Fig. 3.   A: glutathione peroxidase (GPx) mRNA levels in neutrophils, monocytes, monocyte-derived macrophages, and alveolar macrophages. Results, calculated relative to -actin mRNA, are means ± SD of 3 experiments. * P < 0.05 vs. mononuclear cells. B: activity of GPx in neutrophils, monocytes, and monocyte-derived macrophages. * P < 0.05 vs. other cells.

View larger version (10K): [in this window] [in a new window]   Fig. 4.   Cellular glutathione content in neutrophils, monocytes, and monocyte-derived macrophages after 72 h. Results are means ± SD of 4-6 separate experiments. * P < 0.05 vs. neutrophils.

Cell Viability and Cellular Energy State

Cell viability was investigated by trypan blue staining and by following changes in the nucleotide catabolism in cells prelabeled with [¹⁴C]adenine. In agreement with previous studies, only very few, if any, neutrophils survived for 72 h, whereas monocytes maintained their viability for 120 h. The levels of high-energy nucleotides in freshly isolated neutrophils and monocytes were 75-83% of total counts when assessed from the adenine nucleotide pool of the prelabeled cells. The high-energy nucleotides were progressively catabolized within 72 h in neutrophils but remained essentially unchanged in monocytes (Fig. 5). These values, however, overestimate the energy state and the viability of the whole cell population, since the nucleotide levels can be assessed only in viable cells that had incorporated [¹⁴C]adenine. Experiments with BSO-pretreated neutrophils (6-h incubation) indicated that BSO caused a significant depletion of the cellular high-energy nucleotides from 81.5 to 54.6% (n = 2-3) in 6 h. Also BCNU, which inhibits GR, was toxic only to neutrophils, as assessed by nucleotide depletion (data not shown).

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Adenine nucleotide depletion (A) and purine catabolite accumulation (B) in neutrophils (open bars) and monocytes (filled bars) during in vitro culture. Results are means ± SD from 3 separate experiments and expressed as percentage of total soluble radioactivity (counts/min, cpm) in nucleotides (A) or catabolic products (hypoxanthine, xanthine, and uric acid; B). * P < 0.05 vs. corresponding value of neutrophils cultured for 12 h;  P < 0.05 vs. corresponding value of neutrophils cultured for 24 h;  no viable cells available for these experiments.

Effect of FMLP and Exogenous H₂O₂ Exposure on Cell Injury

To compare oxidant sensitivity of these phagocytic cells, they were exposed to FMLP, which activates cell membrane NADPH oxidase. Given that various exogenous stimulators such as FMLP cause a variable response in different phagocytes because of different concentrations of the receptors on the cell surface (9), the cells were also exposed to a defined concentration of H₂O₂, a commonly used and well-characterized oxidant. FMLP caused a significant decrease in cell nucleotides and an accumulation of their catabolic products in the medium of neutrophils, but it did not have any effect on monocytes or monocyte-derived macrophages (Fig. 6). Exposure to 100 µM H₂O₂ for 4 h caused a significant depletion of adenine nucleotides and an accumulation of catabolic products in monocytes and in monocyte-derived macrophages, but not in neutrophils (Fig. 7). When neutrophils were exposed to 500 µM H₂O₂ for 4 h (n = 2-3), no significant adenine nucleotide depletion could be observed, the levels being 76.5% in the control cells and 68.5% in the exposed cells. In mononuclear cells the higher concentration further potentiated the injury, the cell nucleotide levels being 49.1 and 51.5% in monocytes and monocyte-derived macrophages, respectively. Thus neutrophils were more resistant than monocytes to exogenous oxidant exposure. In contrast to the finding that GSH depletion shortened the survival of freshly isolated neutrophils, it had no effect on the resistance of the cells against FMLP or H₂O₂.

View larger version (14K): [in this window] [in a new window]   Fig. 6.   Adenine nucleotide depletion (A) and purine catabolite accumulation (B) in control (open bars) and FMLP-exposed (107 M for 2 h; filled bars), freshly isolated neutrophils, monocytes, and monocyte-derived macrophages cultured for 72 h. Results are means ± SD from 3-6 separate experiments and expressed as percentage of total soluble radioactivity in nucleotides (A) or catabolic products (hypoxanthine, xanthine, and uric acid; B). * P < 0.05 vs. control cells.

View larger version (16K): [in this window] [in a new window]   Fig. 7.   Adenine nucleotide depletion (A) and purine catabolite accumulation (B) in control (open bars) and H2O2-exposed (100 µM for 4 h; filled bars) neutrophils, monocytes, and monocyte-derived macrophages cultured for 72 h. Results are means ± SD from 3-6 separate experiments and expressed as percentage of total soluble radioactivity in nucleotides (A) or catabolic products (hypoxanthine, xanthine, and uric acid; B). * P < 0.05 vs. control cells.

	DISCUSSION

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Activated inflammatory cells generate superoxide by NADPH oxidase on the cell membrane (46, 48) but also in intracellular granules not derived from the plasma membrane (32). The balance between oxidant production and antioxidant defense may have an important role in the phagocytic function and prevention of oxidative damage during phagocytosis. Our results confirm that the most important antioxidant enzymes are expressed in human neutrophils, monocytes, monocyte-derived macrophages, and alveolar macrophages and that differences in their antioxidant profiles may have profound effects on the oxidant resistance of these cells. Neutrophils lost their viability and high-energy nucleotide pool most rapidly, and they contained the lowest levels of GSH and GPx. On the other hand, neutrophils were the most resistant cell type against acute exogenous H₂O₂ exposure and contained the highest levels of Cat mRNA and activity, which may explain this resistance.

Previous studies have investigated SODs during in vitro differentiation of monocytes and HL-60 cells but have not compared their activity in various inflammatory cells or investigated their significance. Our results are in agreement with previous studies showing induction of Mn SOD during the differentiation of monocytes (3, 39). The data on the mRNA levels suggest transcriptional activation of Mn SOD as the mechanism of increased activity. However, previous and present findings on the induction of Mn SOD during monocyte differentiation in vitro cannot be extrapolated to the situation in vivo, since these changes may occur because of cell adherence (53). The levels of Mn SOD in freshly isolated cells are probably similar to the activities in vivo, since previous studies have shown that the activities of these enzymes remain stable during the first hours ex vivo (28).

Mn SOD is induced by inflammatory mediators (endotoxin, tumor necrosis factor-, and interleukin-1) in a number of cells (30, 55). It is also induced in inflammatory cells by cytokines (41, 54) and during granuloma formation in vivo (35). Thus the induction of Mn SOD may play an important role in the resistance of mononuclear cells during inflammation and oxidant stress. A previous study showed no induction of Mn SOD by tumor necrosis factor- in human neutrophils (27), whereas another study showed elevation of the Mn SOD immunoreactive protein by human bronchial epithelial cell-conditioned medium in human neutrophils (16). The present study showed higher relative levels of mRNA, but not the activity of Mn SOD, in neutrophils. The higher mRNA levels may be associated with the activation of these cells during in vitro isolation (27, 42). Discrepancies between the mRNA levels and activities may be related to differences in mRNA stability or translational efficiency of various cells (14, 20). Whether Mn SOD is induced in pathological conditions and contributes to the survival of these cells during inflammation or infection is unclear.

Cat is mainly localized in the peroxisomes of macrophages (18, 43), but it is also found in the cytosol of human neutrophils and alveolar macrophages (7, 43). The present study showed that neutrophils contained the highest Cat activity of all phagocytes. This high activity may have a profound role in the proper phagocytosis during the respiratory burst and oxidant stress of activated neutrophils. The importance of Cat is further supported by the differences in the Michaelis-Menten constant (K_m) of Cat and GPx for H₂O₂, since the K_m for Cat is remarkably higher than the K_m for GPx (26), which also suggests that Cat scavenges H₂O₂ efficiently at high concentrations. We and others have also shown that alveolar macrophages consume exogenous H₂O₂ mainly by a Cat-dependent pathway (38, 43). It is also possible that a mechanism other than Cat may explain the oxidant resistance of neutrophils, but the role of Cat is probably the most important.

GSH metabolism may also play an important role in cell viability and protection against exogenous oxidants of inflammatory cells. This is supported by previous studies showing that phagocytes derived from patients with genetic deficiency of GSH synthase or GR are rapidly damaged during phagocytosis (47, 52) or if the reduction of oxidized GSH is inhibited (15). One previous study has compared the activities of the enzymes of the GSH cycle in neutrophils and monocytes and showed very similar activities of GPx and GR in these cells (56). In contrast, the present study showed that the mRNA and specific activity of GPx, as well as GSH levels, were lower in neutrophils than in monocytes. Lower concentrations of GPx and GSH may contribute to the short life span of neutrophils, since GSH is rapidly decreased in freshly isolated neutrophils (17, 40). It has to be emphasized, however, that cellular GSH is sensitive to oxidizing agents, and GSH metabolism is different in cells of various origins (19). Because the methods of cell isolation were not similar, the interpretation of GSH levels in vivo must be done with caution. The present study also showed that the deterioration of cellular high-energy nucleotides was most prominent in BSO-pretreated neutrophils and that BCNU, which inhibits GR, was toxic only to neutrophils. Although GSH depletion shortened the survival of freshly isolated neutrophils, it had no effect on the viability or energy state of oxidant-exposed inflammatory cells.

We conclude that human inflammatory cells have different antioxidant profiles. Monocytes have higher levels of GSH and enzymes of the GSH cycle, whereas Cat activity is significantly lower in monocytes than in neutrophils. High Cat activity of neutrophils may explain their high resistance to exogenous H₂O₂, whereas low GSH content and GSH-related enzymes may account for the poor survival of human neutrophils.

	ACKNOWLEDGEMENTS

This study was supported by grants from the Finnish Anti-Tuberculosis Association Foundation and the Emil Aaltonen Foundation.

	FOOTNOTES

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: P. Pietarinen-Runtti, Research Laboratory, Hospital for Children and Adolescents, University of Helsinki, PO Box 281, FIN-00029 Helsinki, Finland.

Received 15 June 1999; accepted in final form 3 September 1999.

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