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The Journal of Immunology, 1998, 161: 4960-4967.
Copyright © 1998 by The American Association of Immunologists

Dexamethasone But Not Indomethacin Inhibits Human Phagocyte Nicotinamide Adenine Dinucleotide Phosphate Oxidase Activity by Down-Regulating Expression of Genes Encoding Oxidase Components1

Antonio Condino-Neto*, Constance Whitney{dagger} and Peter E. Newburger2,{dagger}

* Center for Investigation in Pediatrics, State University of Campinas Medical School, Campinas, Brazil; and {dagger} Department of Pediatrics and the Cancer Center, University of Massachusetts Medical Center, Worcester, MA 01655


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We investigated the effects of dexamethasone or indomethacin on the NADPH oxidase activity, cytochrome b558 content, and expression of genes encoding the components gp91-phox and p47-phox of the NADPH oxidase system in the human monocytic THP-1 cell line, differentiated with IFN-{gamma} and TNF-{alpha}, alone or in combination, for up to 7 days. IFN-{gamma} and TNF-{alpha}, alone or in combination, caused a significant up-regulation of the NADPH oxidase system as reflected by an enhancement of the PMA-stimulated superoxide release, cytochrome b558 content, and expression of gp91-phox and p47-phox genes on both days 2 and 7 of cell culture. Noteworthy was the tremendous synergism between IFN-{gamma} and TNF-{alpha} for all studied parameters. Dexamethasone down-regulated the NADPH oxidase system of cytokine-differentiated THP-1 cells as assessed by an inhibition on the PMA-stimulated superoxide release, cytochrome b558 content, and expression of the gp91-phox and p47-phox genes. The nuclear run-on assays indicated that dexamethasone down-regulated the NADPH oxidase system at least in part by inhibiting the transcription of gp91-phox and p47-phox genes. Indomethacin inhibited only the PMA-stimulated superoxide release of THP-1 cells differentiated with IFN-{gamma} and TNF-{alpha} during 7 days. None of the other parameters was affected by indomethacin. We conclude that dexamethasone down-regulates the NADPH oxidase system at least in part by inhibiting the expression of genes encoding the gp91-phox and p47-phox components of the NADPH oxidase system.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Phagocytes contain a membrane-associated nicotinamide adenine dinucleotide phosphate (NADPH)3 oxidase that produces superoxide and other reactive oxygen intermediates responsible for microbicidal, tumoricidal, and inflammatory activities (1, 2). Defects in oxidase activity in chronic granulomatous disease (CGD) lead to severe, life-threatening infections that demonstrate the prime importance of the oxygen-dependent microbicidal system in host defense (3, 4). However, the generation of toxic oxygen species by phagocytes is also instrumental to the tissue damage of diverse conditions, including infection, ischemic injury, arthritis, and other chronic inflammatory and autoimmune disorders (5, 6), and may be contributory to mutation and carcinogenesis (7).

The enzyme system responsible for superoxide generation forms a small transmembrane electron transport system that results in the oxidation of NADPH on the cytoplasmic surface and the generation of superoxide on the outer surface of the membrane. The terminal electron donor to oxygen is a unique, low midpoint potential, flavocytochrome b (8, 9) located primarily in the plasma membrane (10). It is a heterodimer composed of a 91-kDa glycoprotein (termed gp91-phox, for glycoprotein (91 kDa) of phagocyte oxidase) and a 22-kDa polypeptide (p22-phox) (11). The genes for gp91-phox and p22-phox are the sites of mutations responsible for, respectively, the X-linked and one of the autosomal forms of CGD (12, 13). Activation of the NADPH oxidase complex from a resting state to full superoxide-generating activity requires the chemical modification and translocation of additional subunits from the cytosol to the oxidase complex on the cell membrane (14, 15, 16). Two such polypeptides, p47-phox and p67-phox, have been identified and their genes cloned (17, 18). Deficiencies in these two components account for most cases of autosomal recessive CGD (12, 13, 19). In the initial stages of activation, p47-phox undergoes phosphorylation at multiple serine residues in the C-terminal sequence (20). Low m.w. G proteins associated with the oxidase include Rac2, which translocates with the cytosolic oxidase proteins, and Rap1, which closely associates with the p22-phox component in the membrane (21). They probably help to stabilize assembly and regulate activity of the oxidase (22, 23). A newly identified and cloned cytosolic component of the oxidase, p40-phox, associates with p67-phox (24, 25), but definition of its role in oxidase activity awaits further investigation. The gp91-phox and p22-phox genes undergo parallel induction by various cytokines, including IFN-{gamma}, in monocyte-derived macrophages and granulocytes (26, 27).

Anti-inflammatory agents are widely used in clinical medicine and are claimed to protect patients against tissue damage during inflammation at least in part by inhibiting the respiratory burst of phagocytes. Glucocorticoids are the most clinically effective treatment for many inflammatory diseases and provide, in a way, nature’s remedy for inflammation (28, 29). Glucocorticoids have been proven to inhibit superoxide production by phagocytes in a number of experimental models (30, 31, 32, 33, 34, 35, 36, 37), but the contribution of gene regulation to this process remains unknown. Glucocorticoid receptors may interact with transcription factors, including activating protein-1 (38, 39, 40) or NF-{kappa}B (41) to down-regulate gene expression. Glucocorticoids can also up-regulate the expression of the specific NF-{kappa}B inhibitor to decrease its transcriptional induction of enzymes and cytokines associated with inflammation (42, 43). Thus, glucocorticoids may control inflammation by inhibiting several aspects of the inflammatory process through regulation of gene transcription.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are a heterogeneous group of compounds (44). Besides their classical inhibitory effect on the biosynthesis and release of PGs (45, 46, 47), NSAIDs have recently proven to interfere with the transcriptional activation of heat shock factor-1 DNA binding activity (48), and to induce heat shock protein-70 synthesis (49). NSAIDs also interfere with phagocyte NADPH oxidase activity in several experimental models (50, 51, 52, 53). However, the contribution of gene regulation to this process also remains unknown.

Our aim was to investigate the effect of glucocorticoids and NSAIDs on phagocyte NADPH oxidase activity, cytochrome b558 content, and the expression of genes encoding the components gp91-phox and p47-phox of the NADPH oxidase system in human cytokine-differentiated monocytic THP-1 cells. Considering that NADPH oxidase activity does not strictly correlate with the amount of any single component of the NADPH oxidase system regardless of the superoxide release stimulator (54, 55, 56), we focused our experimental approach on the expression of gp91-phox and p47-phox genes, which are the most highly regulated components of the NADPH oxidase system and the most frequent sites for mutations that lead to chronic granulomatous disease (3).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture conditions and experimental protocol

THP-1 cells (57, 58) were cultured in RPMI 1640 complete medium supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 50 µM 2-ME at 37°C in a humidified 5% CO2 atmosphere with IFN-{gamma} (100 U/ml) and TNF-{alpha} (1000 U/ml), alone or in combination, for up to 7 days. All reagents were endotoxin free (<10 pg/ml as tested by Limulus amebocyte lysate assay). As indicated, THP-1 cells received either dexamethasone (0.1–1.0 µM) or indomethacin in a conventional cyclo-oxygenase blocking concentration (10–20 µM) (45, 46, 47) 1 day before starting the cytokine-induced differentiation process to allow intracellular accumulation and functional activity before cytokine stimulation (38, 39, 40, 41, 48, 49, 59, 60). We chose dexamethasone as a standard laboratory representative of the glucocorticoid agents and indomethacin as an archetypal example of a NSAID. Indomethacin was preferred over other NSAIDs because it is a very well-characterized nonspecific cyclo-oxygenase inhibitor, besides being widely used in clinical medicine (61, 62). Cell counts and viability, monitored on a daily basis, were always >80%.

Superoxide release

Superoxide release was assessed by a modified superoxide dismutase (SOD) inhibitable cytochrome c reduction assay (63). Briefly, THP-1 cells were cultured in six-well polystyrene plates (1 x 106 cells/well) with IFN-{gamma} (100 U/ml) and TNF-{alpha} (1000 U/ml), alone or in combination, for up to 7 days in the presence of dexamethasone or indomethacin as described above. On the day of the experiment (day 2 or 7), the plates were centrifuged, the supernatant was removed, and the cytokine-differentiated THP-1 cells were incubated in HBSS (without phenol red) containing cytochrome c (50 µM) and the required cytokines for 1 h at 37°C in a humidified 5% CO2 atmosphere. Half the wells received SOD (60 U/ml) at the beginning of the incubation. In another set of identical plates, PMA (30 nM) was used only during this brief incubation period as an activator of superoxide release. After incubation, all plates were placed on ice, and the other half of the wells received SOD (60 U/ml). The plates were centrifuged again, and the absorbance of the supernatants was monitored at 550 nm. The amount of superoxide released was calculated using an extinction coefficient of 0.021 nM-1 cm-1. The results were expressed as nanomoles of superoxide released per 106 cells per hour.

Cytochrome b558 content

Because p22-phox is constitutively expressed but gp91-phox is required for stability of the cytochrome b558 heterodimer, levels of cytochrome b558 served as the most accurate available assay for the latter component (26, 27). Cytochrome b558 was measured by a spectroscopic method designed to avoid the interference of mitochondrial cytochromes or hemoglobin (64). THP-1 cells were cultured with IFN-{gamma} (100 U/ml) and TNF-{alpha} (1000 U/ml) alone or in combination for up to 7 days in the presence of dexamethasone or indomethacin as described above. On the day of the experiment at least 107 viable cells were harvested, washed three times with PBS, and lysed with 2% Triton X-100 in 0.1 M KH2PO4 buffer at pH 7.25 for 30 min on ice. The lysate was centrifuged at 27,000 x g for 30 min at 4°C, and the supernatant was assayed by spectrophotometric scanning (400–600 nm, 750 nm/min). The test sample received 10 µM KCN, 10 µM NaN3, and a few grains of sodium dithionite and was then aerated by dropwise pipetting over 3 min. The spectrum of the aerated sample was stored in the spectrophotometer memory. The sample was reduced again with a second addition of dithionite and rescanned. The resulting difference spectrum, representing reduced second time - aerated after first reduction, was obtained. The amount of cytochrome b558 was estimated from the height of the band at 558 nm, using an extinction coefficient of 21.6 mM-1 cm-1. The total protein concentration of the samples was determined, and the results were expressed as picomoles of cytochrome b558 per milligram of total protein present in the sample.

Gene expression

To assess gene expression, total cell RNA was extracted from THP-1 cells by the guanidine HCl method (65) and analyzed by Northern blots performed according to standard procedures (66) or slot blots prepared according to the instructions of Schleicher & Schuell (Keene, NH) for their Minifold II apparatus. THP-1 cells were cultured with IFN-{gamma} (100 U/ml) and TNF-{alpha} (1000 U/ml) alone or in combination for up to 7 days in the presence of dexamethasone or indomethacin as described above. Hybridization probes were full-length cDNAs for human gp91-phox (67) and p47-phox (18). Procedures for sequential cycles of prehybridization, washes, and filter stripping were performed as described by Gatti et al. (68). Equal loading of lanes was demonstrated by examination of gels after ethidium bromide staining and by rehybridization with a 5.8-kb HindIII restriction fragment of rat 18S ribosomal cDNA (69). Positive control RNA was obtained from differentiated HL-60 cells, and negative control RNA was obtained from HeLa cells (26, 70).

Transcriptional regulation of gp91-phox and p47-phox was assessed by nuclear run-on transcription assays with minor modifications of previously published procedures (71). THP-1 cells were cultured with IFN-{gamma} (100 U/ml) plus TNF-{alpha} (1000 U/ml) for 2 days in the presence or the absence of 1 µM dexamethasone. Based on the changes in expression of both gp91-phox and p47-phox transcripts in THP-1 cells cultured with IFN-{gamma} (100 U/ml) and TNF-{alpha} (1000 U/ml) for 2 days, we applied these conditions to the run-on assays. Briefly, THP-1 nuclei were isolated by cell lysis in 0.05% Nonidet P-40. Freshly prepared nuclei were incubated for 30 min at 30°C in a reaction mixture containing [32P]UTP (250 µCi, 3000 Ci/mmol) in buffer modified from that described by Greenberg et al. (71) by addition of 0.8 mM MnCl2. Newly synthesized RNA was extracted by the guanidine HCl method (65). Equal amounts of incorporated label from each group (1–2 x 107 cpm) were then hybridized to saturating amounts of cDNA probes, immobilized on filters by slot blotting. The probes used in these experiments included cDNAs for the genes gp91-phox (67) and p47-phox (18), a hybridization negative control (plasmid without insert), and a constitutively expressed gene (ß-actin or {alpha}-tubulin) (72).

Hybridization levels in Northern blots and nuclear run-on assays were quantified by PhosphorImager (Molecular Dynamics, Sunnyvale, CA) and analyzed by ImageQuant software (Molecular Dynamics). Hybridization levels in nuclear run-on assays were normalized to a hybridization negative control (plasmid alone) and to constitutively expressed genes (ß-actin or {alpha}-tubulin) and were calculated as relative rates of transcription (27).

Statistics

Descriptive statistics was performed, and the results were represented by boxplots showing the minimum, 25th percentile, median, 75th percentile, and maximum values (73). The Mann-Whitney U test was used for comparison between groups (74); p < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Dexamethasone inhibits NADPH oxidase activity, cytochrome b558 content, and gene expression of gp91-phox and p47-phox in cytokine-differentiated THP-1 cells

Our first step was to assess the NADPH oxidase activity of THP-1 cells differentiated with IFN-{gamma} (100 U/ml) and TNF-{alpha} (1000 U/ml) alone or in combination. Based on our preliminary data (75) we focused our studies on days 2 and 7. Cytokine-differentiated THP-1 cells have a low spontaneous release of superoxide, similar to unstimulated peripheral blood monocytes (results not shown). However, after PMA (30 nM) stimulation, cytokine-differentiated THP-1 cells release significant higher amounts of superoxide on both days 2 and 7 of cell culture compared with THP-1 cells cultured under basal conditions (Fig. 1Go; p < 0.05 in all situations; n = 6). Noteworthy is the tremendous synergism between IFN-{gamma} and TNF-{alpha}.



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  		FIGURE 1. Dexamethasone inhibits the NADPH oxidase activity of cytokine-differentiated THP-1 cells. THP-1 cells differentiated with IFN-{gamma} (100 U/ml) and TNF-{alpha} (1000 U/ml) alone or in combination for 2 days (A) or 7 days (B) show a higher PMA (30 nM)-stimulated superoxide release (#, p < 0.05; n = 6). Note the strong synergism between IFN-{gamma} (100 U/ml) and TNF-{alpha} (1000 U/ml; A and B). Dexamethasone (Dexa) inhibits PMA-stimulated superoxide release of cytokine-differentiated THP-1 cells (*, p < 0.05; n = 6; A and B) depending on the day of cell culture, cytokine combination, and dexamethasone concentration (0.1, 0.5, or 1.0 µM). Results are represented by boxplots showing the minimum, 25th percentile, median, 75th percentile, and maximum values.

 
We simultaneously investigated the effect of dexamethasone (0.1, 0.5, or 1.0 µM) on the NADPH oxidase activity of THP-1 cells differentiated with IFN-{gamma} (100 U/ml) and TNF-{alpha} (1000 U/ml) alone or in combination during 2 or 7 days. Figure 1Go shows the dose-dependent inhibitory effect of dexamethasone on PMA-stimulated superoxide release by THP-1 cells differentiated with these cytokines compared with that by THP-1 cells cultured in the absence of dexamethasone. Dexamethasone at a concentration of 1 µM inhibited PMA-stimulated superoxide release by THP-1 cells on each day examined regardless of the cytokine combination (p < 0.05 in all situations; n = 6). At a 0.5-µM concentration and on the second day of cell culture (Fig. 1GoA), dexamethasone inhibited PMA-stimulated superoxide release only of TNF-{alpha}-differentiated THP-1 cells (p < 0.05; n = 6); however, on the seventh day of cell culture (Fig. 1GoB), 0.5 µM dexamethasone inhibited PMA-stimulated superoxide release by THP-1 cells cultured with any of the cytokines (p < 0.05 in all situations; n = 6). Dexamethasone at a 0.1-µM concentration inhibited PMA-stimulated superoxide release only of THP-1 cells cultured with IFN-{gamma} and TNF-{alpha} for 7 days (Fig. 1GoB; p < 0.05; n = 6). At other points, inhibition of NADPH oxidase activity by dexamethasone was observed, but did not reach statistical significance (p > 0.05; n = 6).

We further assessed the cytochrome b558 content of THP-1 cells differentiated with IFN-{gamma} (100 U/ml) and TNF-{alpha} (1000 U/ml) alone or in combination for 2 or 7 days. Figure 2Go shows that THP-1 cells differentiated with these cytokines have a higher cytochrome b558 content on both days 2 and 7 of cell culture than THP-1 cells cultured under basal conditions (p < 0.05 in all situations; n = 6). Again, a significant synergism between IFN-{gamma} and TNF-{alpha} occurred. Dexamethasone (1 µM) caused a statistically significant reduction in cytochrome b558 content only in THP-1 cells cultured with IFN-{gamma} (100 U/ml) and TNF-{alpha} (1000 U/ml) for 7 days (p < 0.05; n = 3; Fig. 2GoB). At other points, dexamethasone caused a reduction of the cytochrome b558 content of cytokine-differentiated THP-1 cells. This effect, however, was not statistically significant (p > 0.05; n = 3).



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  		FIGURE 2. The effect of dexamethasone on the cytochrome b558 content of cytokine-differentiated THP-1 cells. THP-1 cells differentiated with IFN-{gamma} (100 U/ml) and TNF-{alpha} (1000 U/ml) alone or in combination for 2 days (A) or 7 days (B) have a higher cytochrome b558 content (#, p < 0.05; n = 6). Again, a strong synergism between IFN-{gamma} (100 U/ml) and TNF-{alpha} (1000 U/ml) occurs (A and B). Dexamethasone (Dexa; 1 µM) causes a significant reduction of the cytochrome b558 content in THP-1 cells cultured with IFN-{gamma} (100 U/ml) and TNF-{alpha} (1000 U/ml) for 7 days (*, p < 0.05; n = 3; B). Results are represented by boxplots showing the minimum, 25th percentile, median, 75th percentile, and maximum values.

 
Considering the results showing the inhibitory effects of dexamethasone on the NADPH oxidase activity of THP-1 cells differentiated with cytokines, we extended our investigation to the gene expression level as assessed by Northern blot hybridization (66).

Figure 3Go shows the induction of gp91-phox and p47-phox gene expression in cytokine-differentiated THP-1 cells. IFN-{gamma} (100 U/ml) alone caused median 5-fold (day 2) and 13-fold (day 7) increases in gp91-phox gene expression and 5-fold (day 2) and 4-fold (day 7) increases in p47-phox gene expression. IFN-{gamma} (100 U/ml) combined with TNF-{alpha} (1000 U/ml) caused median 18-fold (day 2) and 51-fold (day 7) increases in gp91-phox gene expression, and 12-fold (day 2) and 20-fold (day 7) increases in p47-phox gene expression. TNF-{alpha} (1000 U/ml) alone caused median 1.5-fold (day 2) and 3-fold (day 7) increases in gp91-phox gene expression and 1.5-fold (day 2) and 2.8-fold (day 7) increases in p47-phox gene expression (p < 0.05 in all situations; n = 3; calculations based on relative gene expression assessed by computer analysis of PhosphorImager data). Figure 3Go also shows the inhibitory effect of dexamethasone (1 µM) on the expression of gp91-phox and p47-phox genes in THP-1 cells differentiated with IFN-{gamma} (100 U/ml) and TNF-{alpha} (1000 U/ml) alone or in combination during 2 or 7 days (p < 0.05; n = 3; calculations based on relative gene expression assessed by computer analysis of PhosphorImager data). It is noteworthy that dexamethasone inhibited approximately 50% of gp91-phox and p47-phox gene expression in THP-1 cells regardless of the cytokine treatment. Furthermore, in the absence of dexamethasone, gp91-phox and p47-phox gene expression in THP-1 cells differentiated with cytokines for 2 or 7 days correlated with their NADPH oxidase activity and cytochrome b content.



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  		FIGURE 3. Dexamethasone inhibits the expression of gp91-phox and p47-phox genes in cytokine-differentiated THP-1 cells. A representative Northern blot experiment is presented showing that IFN-{gamma} (100 U/ml) and TNF-{alpha} (1000 U/ml) alone or in combination induce the expression of gp91-phox and p47-phox genes in THP-1 cells after 2 days (A) or 7 days (B) of cell culture. Note the tremendous synergism between IFN-{gamma} and TNF-{alpha}. Dexamethasone (Dexa; 1 µM) inhibits the expression of both gp91-phox and p47-phox genes in cytokine-differentiated THP-1 cells regardless of the cytokine combination or the day of cell culture (p < 0.05 in all situations; n = 3; calculations based on relative gene expression assessed by computer analysis of PhosphorImager data. C, control; I, IFN-{gamma}; T, TNF-{alpha}.

 
We further investigated the effect of dexamethasone (1 µM) on the transcription rates of gp91-phox and p47-phox genes in nuclei obtained from cytokine-differentiated THP-1 cells, as assessed by nuclear run-on assays (71). Based on changes in the expression of both gp91-phox and p47-phox transcripts in THP-1 cells cultured with IFN-{gamma} (100 U/ml) and TNF-{alpha} (1000 U/ml) for 2 days, we applied the run-on assays to these conditions. As shown in Figure 4Go, the results demonstrate increased transcription rates of the genes encoding gp91-phox and p47-phox in THP-1 cells cultured with IFN-{gamma} (100 U/ml) and TNF-{alpha} (1000 U/ml) for 2 days in parallel with Northern blot experiments. Transcription rates showed, respectively, 5- and 7.5-fold increases (p < 0.05; n = 3; calculations of relative transcription rates normalized to negative control and to rates for the constitutively expressed genes {alpha}-tubulin and ß-actin, assessed by computer analysis of PhosphorImager data). Dexamethasone (1 µM) significantly inhibited the transcription rates of gp91-phox and p47-phox genes in THP-1 cells cultured with IFN-{gamma} and TNF-{alpha} alone, confirming our previous observations for the gene expression studies as assessed by Northern blot hybridization. Transcription of both genes showed median 50% inhibition (p < 0.05; n = 3).



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  		FIGURE 4. Dexamethasone inhibits the transcription of gp91-phox and p47-phox genes in THP-1 cells differentiated with IFN-{gamma} (100 U/ml) and TNF-{alpha} (1000 U/ml). A representative nuclear run-on experiment is presented showing the inhibitory effect of dexamethasone (Dexa; 1 µM) on the transcription rates of gp91-phox and p47-phox genes in THP-1 cells differentiated with IFN-{gamma} (100 U/ml) and TNF-{alpha} (1000 U/ml) for 2 days (p < 0.05; n = 3; calculations of relative transcription rates normalized to the negative control and to rates for the constitutively expressed genes {alpha}-tubulin and ß-actin, assessed by computer analysis of PhosphorImager data).

 
Effect of indomethacin on the NADPH oxidase activity, cytochrome b558 content, and gene expression of gp91-phox and p47-phox in cytokine-differentiated THP-1 cells

Indomethacin (10 or 20 µM) showed a weak inhibitory effect on the NADPH oxidase activity of cytokine-differentiated THP-1 cells (Table IGo). The drug failed to inhibit PMA-stimulated superoxide release by THP-1 cells cultured with cytokines for 2 days (p > 0.05 in all situations; n = 5). However, on the seventh day of cell culture, indomethacin (10 or 20 µM) significantly inhibited PMA-stimulated superoxide release by THP-1 cells cultured with IFN-{gamma} (100 U/ml) and TNF-{alpha} (1000 U/ml; p < 0.05; n = 5).


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  		Table I. The effect of indomethacin on the NADPH oxidase activity and cytochrome b558 content of cytokine-differentiated THP-1 cells1

 
Indomethacin (10 µM) did not have any inhibitory effect on the cytochrome b558 content of cytokine-differentiated THP-1 cells (Table IGo) regardless of the cytokine treatment or the day of cell culture (p > 0.05 in all situations; n = 4). Similarly, indomethacin (10 µM) failed to inhibit the expression of gp91-phox and p47-phox genes in cytokine-differentiated THP-1 cells on day 2 or 7 of cell culture (p > 0.05; n = 3; results not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Macrophages play a central role in inflammation (1, 2). The oxygen-derived metabolites generated by the phagocyte NADPH oxidase provide an important defense mechanism, but also present a risk for tissue damage (5, 6). Cytokines and anti-inflammatory drugs such as glucocorticoids and NSAIDs are important regulators of inflammation (76) at least in part due to their effects on phagocyte oxidant production (30, 31, 32, 33, 34, 35, 36, 37, 50, 51, 52, 53).

Human monocytic THP-1 cells have well-characterized IFN-{gamma} and TNF-{alpha} receptors (77, 78). Our results show that IFN-{gamma} and TNF-{alpha} alone or in combination induce myelomonocytic THP-1 cells to differentiate and express NADPH oxidase activity, cytochrome b558, and gene transcripts for the NADPH oxidase components gp91-phox and p47-phox. Incubation with IFN-{gamma}, TNF-{alpha}, or the highly synergistic combination induced, respectively, 10-, 2-, and 100-fold increases in PMA-stimulated superoxide release by THP-1 cells. The cytochrome b558 content increased, respectively, 2-, 1.5-, and 5-fold. Expression of the gene encoding the cytochrome b558 component gp91-phox increased, respectively, 5-, 1.5-, and 18-fold on day 2 and 13-, 3-, and 51-fold on day 7. Expression of the gene encoding the cytosolic oxidase component p47-phox increased, respectively, 5-, 1.5-, and 12-fold on day 2 and 4-, 2.8-, and 20-fold on day 7. Nuclear run-on assays showed respective 5- and 7.5-fold increases in gp91-phox and p47-phox gene transcription on the second day of THP-1 cell culture with IFN-{gamma} plus TNF-{alpha}.

Despite the widespread use of glucocorticoids, the molecular mechanisms that underlie their therapeutic effects are poorly understood. Among several effects, glucocorticoids are known to inhibit the production and gene expression of many cytokines, including IL-1{alpha}, IL-1ß, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-10, TNF-{alpha}, TNF-ß, IFN-{gamma}, and growth macrophage CSF (79, 80, 81, 82, 83, 84, 85, 86, 87, 88). Glucocorticoids interact with transcription factors, including activating protein-1 (38, 39, 40) and NF-{kappa}B (41), to down-regulate gene expression. Our studies indicate that dexamethasone down-regulated the NADPH oxidase system at least in part by inhibiting the transcription of gp91-phox and p47-phox genes. The molecular mechanisms involved in this process, such as potential interaction of dexamethasone with transcription factors, are under current investigation in our laboratories.

The production of endogenous inhibitors regulating the NADPH oxidase system and the inhibition of the protein kinase C (PKC) pathway by dexamethasone are likely events in our model system that could partially explain the inhibition of PMA-stimulated superoxide release by cytokine-differentiated THP-1 cells. Depending on the cell lineage and experimental conditions, glucocorticoids either inhibit (89, 90) or do not affect the PKC pathway (91, 92). We have focused our investigation on gp91-phox and p47-phox gene expression studies and correlated them with cytochrome b558 content and NADPH oxidase activity. We propose the regulation of NADPH oxidase gene expression by glucocorticoids as an additional new mechanism for the effect of glucocorticoids on phagocyte oxidase activity. Investigation of other endogenous inhibitors or specific inhibition of the PKC pathway in our model system constitutes a major subject for future investigation.

In contrast, studies in both THP-1 cells and other systems have shown that glucocorticoids can also enhance cytokine responses. For example, dexamethasone and IL-1 synergize to stimulate the production of granulocyte CSF in differentiated THP-1 cells (93). The in vitro enhancement of superoxide anion release stimulated by Mycobacterium leprae or Mycobacterium bovis shows responses to lower concentrations of IFN-{gamma} in monocytes from leprosy patients receiving prednisone therapy than in monocytes from healthy subjects or from other leprosy patients (94). TNF-{alpha} increases glucocorticoid-induced transcriptional activity of the glucocorticoid receptor via the glucocorticoid response elements in mouse fibroblasts (95). These phenomena may reflect a general molecular mechanism by which cytokines or glucocorticoids modulate the transcriptional activity of their receptors, providing counter-regulatory mechanisms at the level of their target cells.

All measures demonstrated tremendous synergism between IFN-{gamma} and TNF-{alpha} induction of NADPH oxidase activity and expression of its components. This synergism has also been observed in a different model system (96). The molecular mechanisms for the synergy between IFN-{gamma} and TNF-{alpha} remain to be investigated.

The nuclear run-on assays indicate that the mechanism of up-regulation of the NADPH oxidase system in our model is at least in part transcriptional. The even larger change in steady state levels of the gene transcripts could represent either accumulation of mRNA due to higher levels of production than degradation or active regulation of mRNA stability. Other post-transcriptional mechanisms, such as translational enhancement, calcium mobilization (97), protein phosphorylation (98), G protein activation (99), or phospholipase A2 activation (100), could contribute to the activation of the NADPH oxidase system and hence the quantitative differences between the cytochrome b558 content and the NADPH oxidase activity results.

As might be expected from a multicomponent enzyme system, NADPH oxidase activity does not strictly correlate with the amount of any single component. For example, eosinophils support considerable oxidase activity with low levels of gp91-phox (54), and in phagocytes from patients with variant forms of chronic granulomatous disease, small changes in gp91-phox content induced by IFN-{gamma} produce seemingly disproportionate increases in respiratory burst activity (55, 56).

IFN-{gamma} was a stronger stimulus than TNF-{alpha} for the induction of oxidase gene expression, cytochrome b558 content and superoxide release. The induction of gp91-phox gene expression in THP-1 cells by IFN-{gamma} alone was previously reported by our group (26). THP-1 cells were less responsive to TNF-{alpha} than human peripheral blood monocytes/macrophages (27). The up-regulation of gp91-phox correlated qualitatively, but not quantitatively, with p47-phox. The gp91-phox gene expression increased in a more dramatic way than that of p47-phox. Whether the gp91-phox gene is more important than the p47-phox gene for the up-regulation of the NADPH oxidase system remains to be determined. Indeed, X-linked chronic granulomatous disease has generally a more severe clinical course than the autosomal form of the disease, which is commonly due to defects in p47-phox (101, 102, 103).

Our studies also demonstrate that indomethacin (10 or 20 µM) inhibited PMA-stimulated superoxide release by THP cells differentiated with IFN-{gamma} and TNF-{alpha} for 7 days, but not to the extent caused by dexamethasone. This effect may be attributed in part to the indomethacin inhibition of both cyclo-oxygenase isoforms (61, 62). At lower levels of PMA-stimulated superoxide release by THP-1 cells differentiated with IFN-{gamma} or TNF-{alpha} alone, the indomethacin inhibitory effect on NADPH oxidase activity could not be detected. In addition, indomethacin did not affect the cells’ cytochrome b558 content or expression of the genes encoding gp91-phox and p47-phox. A direct quenching of active oxygen species by indomethacin (104), the influence of cyclo-oxygenase isoforms in our model system, or other interfering cellular mechanisms remain to be determined.

We conclude that dexamethasone inhibited the NADPH oxidase activity of cytokine-differentiated THP-1 cells at least in part by down-regulating the transcriptional expression of genes encoding components of the NADPH oxidase system. Indomethacin inhibited only the NADPH oxidase activity of cytokine-differentiated THP-1 cells. This mechanism of action of glucocorticoids may be clinically relevant to patients suffering from inflammatory diseases due to the harmful effects of the excessive release of oxygen-derived metabolites.


   Footnotes
 
1 This work was supported by Brazil’s Conselho Nacional de Desenvolvimento Científico e Tecnolólogico (Grant 200955/95-0), Fundação de Amparo à Pesquisa do Estado de São Paulo (Grant 96/11666-2), State University of Campinas Medical School (in-house grant), National Institutes of Health Grant AI33346, and an award from the Howard Hughes Medical Institute to the University of Massachusetts Medical School under the Research Resources Program for Medical Schools. Back

2 Address correspondence and reprint requests to Dr. Peter E. Newburger, Department of Pediatrics, University of Massachusetts Medical Center, 373 Plantation St., Worcester, MA 01605. E-mail address: Back

3 Abbreviations used in this paper: NADPH, nicotinamide adenine dinucleotide phosphate, reduced form; CGD, chronic granulomatous disease; gp91-phox, glycoprotein (91 kDa) of phagocyte oxidase; p47-phox, protein (47 kDa) of phagocyte oxidase; NSAID, nonsteroidal anti-inflammatory drug; SOD, superoxide dismutase; PKC, protein kinase C. Back

Received for publication March 17, 1998. Accepted for publication June 25, 1998.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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M. Luengo-Blanco, C. Prando, J. Bustamante, W. C. Aragao-Filho, P. V. S. Pereira, J. Rehder, C. Padden, J.-L. Casanova, P. E. Newburger, and A. Condino-Neto
Essential role of nuclear factor-{kappa}B for NADPH oxidase activity in normal and anhidrotic ectodermal dysplasia leukocytes
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I. V. Ustyugova, L. L. Frost, K. VanDyke, K. M. Brundage, R. Schafer, and J. B. Barnett
3,4-Dichloropropionaniline Suppresses Normal Macrophage Function
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J. Ehrchen, L. Steinmuller, K. Barczyk, K. Tenbrock, W. Nacken, M. Eisenacher, U. Nordhues, C. Sorg, C. Sunderkotter, and J. Roth
Glucocorticoids induce differentiation of a specifically activated, anti-inflammatory subtype of human monocytes
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E. A. Kurt-Jones, L. Mandell, C. Whitney, A. Padgett, K. Gosselin, P. E. Newburger, and R. W. Finberg
Role of Toll-like receptor 2 (TLR2) in neutrophil activation: GM-CSF enhances TLR2 expression and TLR2-mediated interleukin 8 responses in neutrophils
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D. J. Samuelson, M.-B Powell, M. Lluria-Prevatt, and D. F. Romagnolo
Transcriptional activation of the gp91phox NADPH oxidase subunit by TPA in HL-60 cells
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A. Condino-Neto and P. E. Newburger
Interferon-gamma improves splicing efficiency of CYBB gene transcripts in an interferon-responsive variant of chronic granulomatous disease due to a splice site consensus region mutation
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