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Am. J. Respir. Cell Mol. Biol., Volume 24, Number 2, February 2001 164-169

Differential Induction of TNF-alpha and MnSOD by Endotoxin
Role of Reactive Oxygen Species and NADPH Oxidase

Julie E. White and Min-Fu Tsan

Research Service, Stratton Veterans Affairs Medical Center, and Department of Medicine and Center for Cardiovascular Sciences, Albany Medical College, Albany, New York



    Abstract
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Endotoxin (lipopolysaccharide [LPS]) is known to induce the production of tumor necrosis factor (TNF)-alpha and the induction of manganese superoxide dismutase (MnSOD). We have recently demonstrated that induction of TNF-alpha and MnSOD by LPS is mediated through different signal transduction pathways. In the current study, we investigated the role of reactive oxygen species (ROS) in the induction of TNF-alpha and MnSOD messenger RNAs (mRNAs) in human monocytes. Hypoxia (1% O2) inhibited the production of superoxide (O2-) and the induction of MnSOD, but not TNF-alpha , mRNA. Diphenylene iodonium (DPI), a potent inhibitor of the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, had no effect on LPS induction of MnSOD mRNA, whereas it markedly inhibited LPS-induced O2- production. Neither hypoxia nor DPI had any effect on LPS activation of nuclear factor (NF)-kappa B. These results suggest that (1) ROS is important in the induction of MnSOD, but not TNF-alpha , mRNA by LPS, (2) ROS from sources other than NADPH oxidase is involved in LPS induction of MnSOD mRNA, and (3) ROS-mediated LPS induction of MnSOD mRNA is independent of NF-kappa B activation.



    Introduction
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Endotoxin, lipopolysaccharide (LPS) of Gram-negative bacterial cell wall, is well known to activate monocytes and macrophages, leading to the production of proinflammatory cytokines such as tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta (1, 2). These proinflammatory cytokines, depending on the amount produced, are responsible for the immunostimulatory or endotoxic effect of LPS (1). Endotoxin also induces the antioxidant enzyme, manganese superoxide dismutase (MnSOD) in most cell types (4, 5). Evidence suggests that the induction of lung MnSOD may be responsible for LPS-induced protection against pulmonary O2 toxicity (6, 7).

The mechanism by which LPS induces TNF-alpha production by monocytes and macrophages remains incompletely understood. However, considerable evidence suggests that LPS stimulates both TNF-alpha gene transcription and translation, and that it requires the activation of protein tyrosine kinase (PTK)/Ras/Raf-1/MEK/mitogen-activated protein kinase (MAPK) signal transduction pathway as well as nuclear factor-kappa B (NF-kappa B) (8). On the other hand, little is known about the signal transduction pathway involved in the induction of MnSOD by LPS.

We have recently demonstrated that a mutant Escherichia coli LPS lacking myristoyl fatty acid at the 3' R-3- hydroxymyristate position of the lipid A moiety (nonmyristoyl LPS [nmLPS]) (14) retains its full capacity to coagulate limulus amebocyte lysate and markedly stimulates the activation of NF-kappa B and the induction of MnSOD in human monocytes (15). However, in contrast to the wild-type LPS, nmLPS fails to induce significant production of TNF-alpha and does not induce the phosphorylation and nuclear translocation of p42 MAPK (15).

Likewise, inhibitors of PTK markedly inhibit the activation of PTK and MAPK, and the production of TNF-alpha by LPS-treated human monocytes. However, they have no effect on LPS induction of NF-kappa B and MnSOD. In contrast, inhibition of NF-kappa B activation inhibits LPS induction of both TNF-alpha and MnSOD (16). These results suggest that induction of TNF-alpha and MnSOD in human monocytes by LPS are, at least in part, regulated through different signal transduction pathways. Although PTK and MAPK are essential for the production of TNF-alpha , they are not necessary for the induction of MnSOD by LPS. On the other hand, although activation of NF-kappa B alone is insufficient for the induction of TNF-alpha , it is necessary for the induction of both TNF-alpha and MnSOD by LPS (15, 16).

Considerable evidence suggests that reactive oxygen species (ROS) may play an important role in the induction of MnSOD (17). In the current study, we investigated the role of ROS in the LPS induction of TNF-alpha and MnSOD. We demonstrated that exposure of human monocytes to hypoxia (1% O2) markedly reduced LPS-induced superoxide (O2-) production. Hypoxic exposure also reduced LPS induction of MnSOD, but not TNF-alpha , messenger RNA (mRNA). Diphenylene iodonium (DPI), a potent inhibitor of the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (20), markedly reduced LPS-induced O2- production. However, it had no effect on the LPS induction of TNF-alpha or MnSOD mRNA. Neither hypoxia nor DPI had any effect on the activation of NF-kappa B by LPS. These results suggest that ROS is important in the LPS induction of MnSOD, but not TNF-alpha , mRNA and that ROS from sources other than NADPH oxidase is involved in the LPS induction of MnSOD mRNA.


    Materials and Methods
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Materials

Protein-free endotoxin (LPS from JM83 E. coli K-12, rough form) was kindly provided by John E. Somerville of Bristol-Myers Squibb Pharmaceutical Research Institute (Princeton, NJ). DPI, ferricytochrome c, phorbol myristate acetate (PMA), and copper-zinc superoxide dismutase (CuZnSOD) (from bovine erythrocyte) were obtained from Sigma Chemical Co. (St. Louis, MO). CuZnSOD had a specific activity of 4,400 U/mg protein as determined according to the method of McCord and Fridovich (21). Stock solutions of DPI and PMA were made in dimethyl sulfoxide (DMSO). The concentrations of DMSO in the final incubation medium were no more than 0.05%. In each case, equivalent amounts of DMSO were used as controls.

Isolation of Human Monocytes

Human mononuclear cells were isolated from the venous blood of normal volunteers (after explaining the nature and possible risks of the studies and obtaining informed consents) using Isolymph (Gallard-Schlesinger Industries Inc., Carle Place, NY) as described previously (15). Cells (5 × 106/ml in RPMI 1640 culture medium plus antibiotics and 10% autologous serum) were allowed to adhere onto tissue culture plates for 2 h. Adherent cells that consisted of approximately 90% monocytes as judged by the nonspecific esterase staining were used for the current studies. The viability of the cells was more than 95% as determined by trypan blue dye exclusion. All experiments were performed in the presence of 10% serum.

Exposure of Monocytes to Hypoxia

Adherent monocytes, after treatment as described in subsequent text, were immediately placed in an oxygen chamber (Bellco Glass, Inc., Vineland, NJ), flushed for 15 min with a mixture of 1% O2, 5% CO2, and 94% N2 (hypoxia), and incubated at 37°C for 1 to 3 h. For normoxia control, cells were exposed to a mixture of 5% CO2 plus room air. The PO2 level and pH of the incubation medium were measured using a blood gas analyzer (ABL3 Acid Base Radiometer, Copenhagen, Denmark).

Measurements of O2- Production

This was performed using superoxide dismutase (SOD)-inhibitable reduction of ferricytochrome c as described previously (22). Briefly, adherent monocytes in a final volume of 1 ml Hanks' balanced salt solution containing 10% human serum and 20 µM cytochrome c, with or without 200 U/ml CuZnSOD, were treated with or without LPS (10 ng/ml or 1 µg/ml), or PMA (0.1 µg/ml) at 37°C for 15 min to 2 h. The incubation was carried out in either normoxic or hypoxic conditions as described previously. In some experiments, cells were pretreated with DPI (0.5 to 5 µM) or DMSO (0.05%, the concentration of DMSO present in 5 µM DPI, as vehicle control) for 30 min before treatment with LPS.

At the end of incubation, aliquots (100 µl) of supernates were placed in a 96-well microtiter plate and the absorbance at 550 nm was determined using a microplate spectrophotometer (SpectraMAX Plus; Molecular Devices Corp., Sunnyvale, CA). The difference of absorbance at 550 nm in the presence and absence of SOD was taken as a measurement of O2-. The amount of O2- produced was calculated using an extinction coefficient of Delta E550 nm = 2.1 × 104 M-1cm-1.

Northern Blot Analysis of TNF-alpha and MnSOD mRNAs

This was performed as described previously (15, 16). Briefly, adherent monocytes in RPMI 1640 culture medium plus 10% serum were treated with or without LPS (10 ng/ml or 1 µg/ml) at 37°C for 3 h under normoxic or hypoxic conditions as described previously. In some experiments, monocytes were pretreated with or without DPI (1 µM) or DMSO (0.01%, as vehicle control) for 30 min before treatment with LPS.

The total cellular RNA was then isolated by the single step method of Chomczynski and Sacchi (23) using RNeasy Mini Kit (Qiagen Inc., Chatsworth, CA). For Northern blots, denatured RNA samples (3 µg/lane) were electrophoresed in 1.2% agarose gels, transferred to nylon membrane (Genescreen plus; New England Nuclear, Boston, MA) by capillary blotting, and stained with methylene blue to visualize the quality and size of 18S and 28S ribosomal RNA species. The membrane was then prehybridized as described previously (24). Hybridization was carried out with 100 µg/ml denatured salmon testis DNA and complementary DNA (cDNA) probes that had been labeled by random hexanucleotide priming (GIBCO/BRL, Gaithersburg, MD) to a specific activity of > 109 cpm/µg DNA. The cDNA probes used included human TNF-alpha and human MnSOD (American Type Culture Collection, Rockville, MD). After washing, autoradiographs were obtained and radioactive signals were quantified using a computing densitometer (Molecular Dynamics, Sunnyvale, CA).

Electrophoresis Mobility Shift Assay for NF-kappa B

This was performed as described previously (15, 16). Adherent monocytes in RPMI 1640 culture medium plus 10% serum were pretreated with or without DPI (1 µM) or DMSO (0.01%) for 30 min. They were then treated with or without 1 µg/ml LPS for 1 h at 37°C. The nuclear extracts were obtained according to Osnes and coworkers (25). For the electrophoresis mobility shift assay (EMSA), 2-µg nuclear proteins were incubated for 30 min at room temperature with ~ 100,000 cpm (5 ng) of an oligonucleotide containing NF-kappa B consensus sequence (5'-AGT TGA GGG GAC TTT CCC AGG C-3') that had been 5'-end labeled with [gamma -32P]adenosine triphosphate using T4 polynucleotide kinase (Promega, Madison, WI). Competition was carried out using a 100-fold excess of the unlabeled oligonucleotide 10 min before adding the radiolabeled probe. Samples were then electrophoresed in a 6% nondenaturing polyacrylamide gel. Autoradiographs were obtained and radioactive signals were quantified using a computing densitometer.

Statistical Analysis

Data from two groups were compared by a two-tailed t test, and those from more than two groups were compared by one-way analysis of variance with Bonferroni correction for multiple comparison (26) using a commercially available statistical analysis program (SPSS Inc., Arlington, VA). A difference is considered to be significant at P < 0.05.


    Results
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Effect of Hypoxia on LPS Induction of TNF-alpha and MnSOD mRNAs

To determine the role of ROS in the induction of TNF-alpha and MnSOD mRNAs by LPS, we first evaluated the effect of hypoxia because hypoxia is well known to reduce ROS production. We have previously demonstrated that LPS induces both TNF-alpha and MnSOD mRNAs in human monocytes at 3 h after treatment (15). For this reason, the current study was performed at 3 h after treatment with LPS.

The results of a representative Northern blot are shown in Figure 1A. Figures 1B and 1C summarize the effects of hypoxia on LPS induction of TNF-alpha and MnSOD mRNAs. For comparison, levels of TNF-alpha and MnSOD (1 and 4 kb) mRNAs, corrected for RNA loading using 28S ribosomal RNA, in control cells exposed to normoxia were normalized to one relative densitometric unit. Both the membrane CD14-dependent (10 ng/ml) and -independent (1 µg/ml) concentrations of LPS were studied. LPS at 10 ng/ml or 1 µg/ml markedly stimulated the steady-state levels of TNF-alpha and MnSOD mRNAs under normoxic condition. Hypoxia, while it had no effect on LPS induction of TNF-alpha mRNA, markedly reduced LPS-induced increases in levels of MnSOD mRNA. Measurements of pO2 revealed that at 3 h after hypoxic (1% O2) exposure, the pO2 level of the incubation medium was reduced to 70 mm Hg, as compared with 150 mm Hg in normoxia (20% O2)- exposed medium (mean of two experiments). The pH of the medium remained constant (7.35 to 7.45).



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  		Figure 1.   Effect of hypoxia on LPS induction of TNF-alpha and MnSOD mRNAs in human monocytes. Adherent monocytes were treated with or without LPS (10 ng/ml or 1 µg/ml) at 37°C for 3 h under a normoxic (20% O2) or hypoxic (1% O2) condition. Cellular RNAs were extracted and aliquots (3 µg/lane) were subjected to Northern blot analysis for TNF-alpha and MnSOD mRNAs. (A) A representative Northern blot. Lanes 1-3, normoxia; lanes 4-6, hypoxia. Lanes 1 and 4, control; lanes 2 and 5, LPS (10 ng/ml); lanes 3 and 6, LPS (1 µg/ml). The effects of hypoxia (solid bars) were summarized in (B) for TNF-alpha mRNA and (C) for MnSOD mRNA (normoxia, open bars). For comparison, levels of TNF-alpha and MnSOD (1 and 4 kb) mRNAs corrected for RNA loading using 28S ribosomal RNA in control cells exposed to normoxia were normalized to one relative densitometric unit. Results were mean ± SE of six experiments. *P < 0.05 versus normoxia.

Effect of Hypoxia on O2- Production

To determine whether hypoxic exposure in fact reduced ROS production, we measured O2- production by human monocytes under normoxia and hypoxia. As shown in Figure 2, LPS at 10 ng/ml or 1 µg/ml was capable of stimulating the production of O2- by human monocytes under normoxic condition, although to a much smaller extent than PMA (Figure 2A, a representative time course experiment; Figure 2B, a summary of the results).



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  		Figure 2.   Effects of LPS and PMA on O2- production by human monocytes. Adherent monocytes (from 5 × 106 mononuclear cells) were treated with or without LPS (10 ng/ml or 1 µg/ ml) or PMA (0.1 µg/ml) at 37°C for 15 to 120 min under normoxia. At the end of incubation, the amounts of O2- produced were determined using an SOD-inhibitable reduction of cytochrome c. (A) A representative time course experiment. (B) Summary of the effects of LPS (solid bars) and PMA (hatched bars). Control, open bars. Results were mean ± SE of seven experiments. P values, *P < 0.001 versus control; §P > 0.001 versus LPS.

Because LPS at 10 ng/ml and 1 µg/ml had similar effects on the induction of TNF-alpha and MnSOD mRNAs as well as the production of O2- by human monocytes, our subsequent experiments were carried out using 1 µg/ml of LPS. As shown in Figure 3, hypoxia markedly reduced both the basal and LPS-induced O2- production by human monocytes.



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  		Figure 3.   Effects of hypoxia on LPS-induced O2- production by human monocytes. Adherent monocytes (from 5 × 106 mononuclear cells) were treated with or without LPS (1 µg/ml) at 37°C for 60 or 120 min under normoxic (open bars) or hypoxic (solid bars) conditions. The amounts of O2- produced were then determined as described in Figure 2. Results were mean ± SE of five experiments. P values versus normoxia, * < 0.02; ** < 0.01; § < 0.05.

Role of NADPH Oxidase

The results mentioned previously demonstrated that hypoxia reduced LPS-induced O2- production as well as MnSOD mRNA induction, suggesting that ROS might play an important role in the LPS induction of MnSOD mRNA. In order to determine the source of ROS that might be involved in the upregulation of MnSOD mRNA, we studied the effect of DPI, a potent inhibitor of NADPH oxidase (20).

As shown in Figure 4, DPI (0.5 to 5 µM) markedly inhibited O2- production by control, nonstimulated human monocytes, suggesting that NADPH oxidase was responsible for most of the basal O2- production. DPI also markedly inhibited the LPS-induced increase in O2- production. DPI at concentrations ranging from 0.5 to 5 µM appeared to inhibit human monocyte O2- production to similar extents. At these concentrations, DPI was not toxic to human monocytes as judged by trypan blue dye exclusion (data not shown).



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  		Figure 4.   Effects of DPI on LPS-induced O2- production by human monocytes. Adherent monocytes (from 5 × 106 mononuclear cells) were pretreated with DPI (0.5 to 5 µM) or with DMSO (0.05%, as vehicle control) for 30 min. They were then treated with or without LPS (1 µg/ml) at 37°C for 60 min, and the amounts of O2- produced were determined. (A) A representative dose-response experiment. (B) Summary of the effect of DPI (solid bars). Control, open bars. Results were mean ± SE of three experiments. P values versus control, * < 0.01; § < 0.001.

We then determined the effect of DPI on LPS induction of TNF-alpha and MnSOD mRNAs. As shown in Figure 5, DPI at 1 µM had no effect on the steady-state levels of TNF-alpha and MnSOD mRNAs in control (nonstimulated) or LPS-treated human monocytes.



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  		Figure 5.   Effects of DPI on LPS induction of TNF-alpha and MnSOD mRNAs in human monocytes. Adherent monocytes were pretreated with DPI (1 µM) or with DMSO (0.01% as vehicle control) for 30 min. They were then treated with or without LPS (1 µg/ml) at 37°C for 3 h under normoxia. Cellular RNAs were extracted and aliquots (3 µg/lane) were subjected to Northern blot analysis for TNF-alpha and MnSOD mRNAs. (A) A representative Northern blot. Lane 1, control; lane 2, LPS; lane 3, DPI; lane 4, DPI + LPS. The effects of DPI are summarized in (B) for TNF-alpha mRNA and (C) for MnSOD mRNA. For comparison, levels of TNF-alpha and MnSOD (1 and 4 kb) mRNAs corrected for RNA loading using 28S ribosomal RNA in control cells were normalized to 1 relative densitometric unit. Results were mean ± SE of four experiments. *P < 0.001 versus control.

Effect of Hypoxia and DPI on NF-kappa B

We have previously demonstrated that activation of NF-kappa B is necessary for the induction of both TNF-alpha and MnSOD mRNAs by LPS (16). To determine whether the previous observed decrease in LPS induction of MnSOD by hypoxia was due to a reduced activation of NF-kappa B, we studied the effect of hypoxia and DPI. As shown in Figure 6, neither DPI nor hypoxia at concentrations that markedly inhibited O2- production had any effect on LPS activation of NF-kappa B.



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  		Figure 6.   Effects of hypoxia and DPI on LPS activation of NF-kappa B in human monocytes. Adherent monocytes were pretreated with DPI (5 µM) or with DMSO (0.05% as vehicle control) for 30 min, followed by treatment with or without LPS (1 µg/ml) at 37°C for 1 h under normoxic or hypoxic conditions. Nuclear extracts were obtained and probed for NF-kappa B using EMSA. (A) A representative EMSA. Lanes 1-4 and 7-10, normoxia; lanes 5, 6, 11, and 12, hypoxia. Lane 1, control; lane 2, LPS; lane 3, DPI; lane 4, DPI + LPS; lane 5, control; lane 6, LPS; lane 7, control + competition; lane 8, LPS + competition; lane 9, DPI + competition; lane 10, DPI + LPS + competition; lane 11, control + competition; lane 12, LPS + competition. (B) Summary of the effects of hypoxia and DPI. Results were expressed as relative densitometric units of mean ± SE of three experiments.


    Discussion
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

The data presented in the current study demonstrated that hypoxia inhibited the production of O2- and induction of MnSOD, but not TNF-alpha , mRNA by LPS in human monocytes, suggesting that ROS might be involved in LPS induction of MnSOD mRNA. Hypoxia had no effect on LPS activation of NF-kappa B. Thus, the reduced induction of MnSOD mRNA by hypoxic exposure in LPS-treated human monocytes was not mediated by a reduced activation of NF-kappa B.

There has been considerable interest in the role of ROS in MnSOD induction (17, 27). However, most evidence supporting the role of ROS in the induction of MnSOD is derived from the use of antioxidants or exogenous oxidants. These previous studies suffer a number of shortcomings. First, most antioxidants have nonspecific effects other than scavenging oxidants. Second, not all antioxidants with presumed similar specificities against particular species of ROS show similar effects in preventing MnSOD induction. For example, n-acetylcysteine (NAC), but not pyrrolidine dithiocarbamate (PDTC) or vitamin E, prevents TNF-alpha induction of MnSOD mRNA in H441 human pulmonary adenocarcinoma cells (17). Furthermore, in A549 human pulmonary adenocarcinoma cells, NAC induces MnSOD by itself (27). Third, none of the studies actually measures the production of ROS. The role of ROS in MnSOD induction was inferred only from the use of antioxidants (17, 27, 28). Fourth, whereas exogenous oxidants such as H2O2 (17) or high concentrations of O2 (hyperoxia) (29) have been shown to induce MnSOD in a number of cell types, its time course is vastly different from the induction of MnSOD by LPS or TNF-alpha , e.g., 12 h after H2O2 treatment versus less than 3 h after TNF-alpha or LPS treatment (17). Thus, the role of ROS in the induction of MnSOD by LPS or TNF-alpha remains unclear.

To overcome the previously mentioned shortcomings, in the current study we used a short-term exposure to hypoxia and quantified the production of O2- by human monocytes using an assay that was specific for O2- (30). Our results revealed that hypoxia reduced LPS-induced O2- production as well as MnSOD, but not TNF-alpha , mRNA in human monocytes. We have also investigated the effect of antioxidants PDTC and NAC. We have previously reported that PDTC (0.2 mM) markedly inhibits LPS induction of TNF-alpha and MnSOD mRNAs (16). However, unlike hypoxia, PDTC also markedly reduces the activation of NF-kappa B by LPS (16). Presumably, inhibition of NF-kappa B activation by PDTC is responsible for its inhibition of TNF-alpha and MnSOD mRNA induction by LPS. On the other hand, NAC, at a concentration as high as 100 mM, had no effect on LPS activation of NF-kappa B in human monocytes. It inhibited LPS induction of MnSOD only at 100 mM, but not at 50 mM (data not shown). Because of the extremely high concentration (100 mM) of NAC required to inhibit LPS induction of MnSOD, the results were difficult to interpret. These data from PDTC and NAC further demonstrate the previously described difficulties in using antioxidants alone in evaluating the role of ROS.

It has been shown that hypoxia enhances LPS-induced production of TNF-alpha by human monocytes (31). However, this requires a prolonged exposure to hypoxia, e.g., over 16 h. Prolonged exposure (> 12 h) to hypoxia alone has been shown to induce TNF-alpha mRNA in T84 intestinal epithelial cells, in part due to a downregulation of cyclic adenosine monophosphate response element-binding protein expression (32). Prolonged exposure to hypoxia (24 h) also reduces levels of MnSOD mRNA in rat type II alveolar epithelial cells and lung fibroblasts (33). In the current study, we exposed human monocytes to hypoxia for up to 3 h, and there was no change in basal levels of TNF-alpha and MnSOD mRNAs, or in an LPS-induced increase in TNF-alpha mRNA; however, it reduced an LPS-induced increase in MnSOD mRNA. A recent study by Ndengele and colleagues (34) also demonstrated that a brief exposure of RAW 264.7 murine macrophages to 0% O2 (95% N2-5% CO2), while having no effect on an LPS-induced increase in TNF-alpha mRNA, markedly inhibited an LPS-induced increase in IL-1 mRNA.

In an attempt to define the source of ROS that might be involved in the induction of MnSOD mRNA by LPS, we studied the effect of DPI, a potent inhibitor of NADPH oxidase (20). Although it inhibited almost completely LPS-induced increase in O2- production by human monocytes, DPI had no effect on LPS induction of TNF-alpha and MnSOD mRNAs. Thus, the ROS that is involved in LPS induction of MnSOD mRNA is not from NADPH oxidase. Warner and associates (17) also report that inhibition of NADPH oxidase by 4'-hydroxy-3'-methoxy-acetophenone has no effect in TNF-alpha induction of MnSOD mRNA in H441 human pulmonary adenocarcinoma cells.

It should be pointed out that although hypoxia reduces both intracellular and extracellular production of ROS, including O2-, in the current study we used an SOD-inhibitable reduction of cytochrome c for measuring O2- production. Because SOD and cytochrome c do not cross the cell membrane readily, we were primarily measuring O2- that was produced by human monocytes and secreted into the medium. NADPH oxidase is a membrane enzyme (35) and is thus the major source of this extracellular O2-. It is not clear how much of the O2- produced by activated NADPH oxidase contributes to the intracellular pool of ROS.

The previously mentioned observations are consistent with the recent hypothesis that a high level of ROS produced by NADPH oxidase in neutrophils and macrophages is primary for immune defense, whereas a low level of ROS plays an important role in signal transduction (36). Other sources of ROS include the mitochondrial electron transport system, xanthine oxidase, cyclooxygenase, lipoxygenase, and Rac1 (36, 37). We have attempted to measure intracellular ROS spectrofluorometrically using 123-dihydrorhodamine and dichlorofluorecin diacetate without success, in part due to problems inherent to human monocytes (38). Furthermore, these fluorescent probes measure intracellular as well as extracellular ROS (39). Thus, the source and the mechanism by which ROS is involved in the LPS induction of MnSOD mRNA require further investigation.


    Footnotes

Address correspondence to: Min-Fu Tsan, M.D., Ph.D., Research Service (151), Stratton VA Medical Center, 113 Holland Ave., Albany, NY 12208. E-mail: min-fu.tsan{at}med.va.gov

(Received in original form March 13, 2000 and in revised form October 5, 2000).

Acknowledgments: This work was supported by the Medical Research Service, Office of Research and Development, Department of Veterans Affairs.

Abbreviations CuZnSOD, copper-zinc superoxide dismutase; DMSO, dimethyl sulfoxide; DPI, diphenylene iodonium; EMSA, electrophoresis mobility shift assay; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; MnSOD, manganese superoxide dismutase; mRNA, messenger RNA; NAC, n-acetylcysteine; NADPH, reduced nicotinamide adenine dinucleotide phosphate; NF-kappa B, nuclear factor kappa B; O2-, superoxide; PDTC, pyrrolidine dithiocarbamate; PMA, phorbol myristate acetate; PTK, protein tyrosine kinase; ROS, reactive oxygen species; SE, standard error; SOD, superoxide dismutase; TNF, tumor necrosis factor.


    References
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

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