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Dexamethasone But Not Indomethacin Inhibits Human Phagocyte Nicotinamide Adenine Dinucleotide Phosphate Oxidase Activity by Down-Regulating Expression of This information is current as Genes Encoding Oxidase Components of September 27, 2021. Antonio Condino-Neto, Constance Whitney and Peter E. Newburger J Immunol 1998; 161:4960-4967; ; http://www.jimmunol.org/content/161/9/4960 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. 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,† and Peter E. Newburger2†

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-␥ and TNF-␣, alone or in combination, for up to 7 days. IFN-␥ and TNF-␣, alone or in combination, caused a signiﬁcant up-regulation of the NADPH oxidase system as reﬂected by an enhancement of the PMA- Downloaded from 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-␥ and TNF-␣ 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 differen- http://www.jimmunol.org/ tiated with IFN-␥ and TNF-␣ 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. The Journal of Immunology, 1998, 161: 4960–4967.

hagocytes contain a membrane-associated nicotinamide The enzyme system responsible for superoxide generation forms adenine dinucleotide phosphate (NADPH)3 oxidase that a small transmembrane electron transport system that results in the P produces superoxide and other reactive oxygen intermedi- oxidation of NADPH on the cytoplasmic surface and the genera- ates responsible for microbicidal, tumoricidal, and inflammatory tion of superoxide on the outer surface of the membrane. The activities (1, 2). Defects in oxidase activity in chronic granuloma- terminal electron donor to oxygen is a unique, low midpoint po- by guest on September 27, 2021 tous disease (CGD) lead to severe, life-threatening infections that tential, flavocytochrome b (8, 9) located primarily in the plasma demonstrate the prime importance of the oxygen-dependent mi- membrane (10). It is a heterodimer composed of a 91-kDa glyco- crobicidal system in host defense (3, 4). However, the generation protein (termed gp91-phox, for glycoprotein (91 kDa) of phago- of toxic oxygen species by phagocytes is also instrumental to cyte oxidase) and a 22-kDa polypeptide (p22-phox) (11). The the tissue damage of diverse conditions, including infection, genes for gp91-phox and p22-phox are the sites of mutations re- ischemic injury, arthritis, and other chronic inflammatory and sponsible for, respectively, the X-linked and one of the autosomal autoimmune disorders (5, 6), and may be contributory to mu- forms of CGD (12, 13). Activation of the NADPH oxidase com- tation and carcinogenesis (7). plex from a resting state to full superoxide-generating activity re- quires the chemical modification and translocation of additional subunits from the cytosol to the oxidase complex on the cell membrane (14–16). Two such polypeptides, p47-phox and p67-phox, *Center for Investigation in Pediatrics, State University of Campinas Medical School, have been identified and their genes cloned (17, 18). Deficiencies Campinas, Brazil; and †Department of Pediatrics and the Cancer Center, University of Massachusetts Medical Center, Worcester, MA 01655 in these two components account for most cases of autosomal re- Received for publication March 17, 1998. Accepted for publication June 25, 1998. cessive CGD (12, 13, 19). In the initial stages of activation, p47- phox undergoes phosphorylation at multiple serine residues in the The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance C-terminal sequence (20). Low m.w. G proteins associated with with 18 U.S.C. Section 1734 solely to indicate this fact. the oxidase include Rac2, which translocates with the cytosolic 1 This work was supported by Brazil’s Conselho Nacional de Desenvolvimento Ci- oxidase proteins, and Rap1, which closely associates with the p22- entı´fico e Tecnolo´logico (Grant 200955/95-0), Fundaçaõ de Amparo a`Pesquisa do Estado de Saõ Paulo (Grant 96/11666-2), State University of Campinas Medical phox component in the membrane (21). They probably help to School (in-house grant), National Institutes of Health Grant AI33346, and an award stabilize assembly and regulate activity of the oxidase (22, 23). A from the Howard Hughes Medical Institute to the University of Massachusetts Med- newly identified and cloned cytosolic component of the oxidase, ical School under the Research Resources Program for Medical Schools. p40-phox, associates with p67-phox (24, 25), but definition of its 2 Address correspondence and reprint requests to Dr. Peter E. Newburger, Department of Pediatrics, University of Massachusetts Medical Center, 373 Plantation St., role in oxidase activity awaits further investigation. The gp91-phox Worcester, MA 01605. E-mail address: [email protected] and p22-phox genes undergo parallel induction by various cyto- 3 Abbreviations used in this paper: NADPH, nicotinamide adenine dinucleotide phos- kines, including IFN-␥, in monocyte-derived macrophages and phate, reduced form; CGD, chronic granulomatous disease; gp91-phox, glycoprotein granulocytes (26, 27). (91 kDa) of phagocyte oxidase; p47-phox, protein (47 kDa) of phagocyte oxidase; NSAID, nonsteroidal anti-inflammatory drug; SOD, superoxide dismutase; PKC, pro- Anti-inflammatory agents are widely used in clinical medicine tein kinase C. and are claimed to protect patients against tissue damage during

inflammation at least in part by inhibiting the respiratory burst of of the incubation. In another set of identical plates, PMA (30 nM) was used phagocytes. Glucocorticoids are the most clinically effective treat- only during this brief incubation period as an activator of superoxide re- ment for many inflammatory diseases and provide, in a way, na- lease. 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 ture’s remedy for inflammation (28, 29). Glucocorticoids have the absorbance of the supernatants was monitored at 550 nm. The amount been proven to inhibit superoxide production by phagocytes in a of superoxide released was calculated using an extinction coefficient of number of experimental models (30–37), but the contribution of 0.021 nMϪ1 cmϪ1. The results were expressed as nanomoles of superoxide 6 gene regulation to this process remains unknown. Glucocorticoid released per 10 cells per hour. receptors may interact with transcription factors, including acti- ␬ vating protein-1 (38–40) or NF- B (41) to down-regulate gene Cytochrome b content expression. Glucocorticoids can also up-regulate the expression of 558 the specific NF-␬B inhibitor to decrease its transcriptional induc- Because p22-phox is constitutively expressed but gp91-phox is required for stability of the cytochrome b heterodimer, levels of cytochrome b tion of enzymes and cytokines associated with inflammation (42, 558 558 served as the most accurate available assay for the latter component (26, 43). Thus, glucocorticoids may control inflammation by inhibiting 27). Cytochrome b558 was measured by a spectroscopic method designed to several aspects of the inflammatory process through regulation of avoid the interference of mitochondrial cytochromes or hemoglobin (64). gene transcription. THP-1 cells were cultured with IFN-␥ (100 U/ml) and TNF-␣ (1000 U/ml) Nonsteroidal anti-inflammatory drugs (NSAIDs) are a hetero- 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 geneous group of compounds (44). Besides their classical inhibi- 107 viable cells were harvested, washed three times with PBS, and lysed

tory effect on the biosynthesis and release of PGs (45–47), with 2% Triton X-100 in 0.1 M KH2PO4 buffer at pH 7.25 for 30 min on NSAIDs have recently proven to interfere with the transcriptional ice. The lysate was centrifuged at 27,000 ϫ g for 30 min at 4°C, and the Downloaded from activation of heat shock factor-1 DNA binding activity (48), and to supernatant was assayed by spectrophotometric scanning (400–600 nm, 750 nm/min). The test sample received 10 ␮M KCN, 10 ␮M NaN , and a induce heat shock protein-70 synthesis (49). NSAIDs also interfere 3 few grains of sodium dithionite and was then aerated by dropwise pipetting with phagocyte NADPH oxidase activity in several experimental over 3 min. The spectrum of the aerated sample was stored in the spec- models (50–53). However, the contribution of gene regulation to trophotometer memory. The sample was reduced again with a second ad- this process also remains unknown. dition of dithionite and rescanned. The resulting difference spectrum, rep- Ϫ Our aim was to investigate the effect of glucocorticoids and resenting reduced second time aerated after ﬁrst reduction, was obtained. http://www.jimmunol.org/ The amount of cytochrome b558 was estimated from the height of the band NSAIDs on phagocyte NADPH oxidase activity, cytochrome b558 at 558 nm, using an extinction coefﬁcient of 21.6 mMϪ1 cmϪ1. The total content, and the expression of genes encoding the components protein concentration of the samples was determined, and the results were

gp91-phox and p47-phox of the NADPH oxidase system in human expressed as picomoles of cytochrome b558 per milligram of total protein cytokine-differentiated monocytic THP-1 cells. Considering that present in the sample. NADPH oxidase activity does not strictly correlate with the amount of any single component of the NADPH oxidase system Gene expression regardless of the superoxide release stimulator (54–56), we focused our experimental approach on the expression of gp91-phox To assess gene expression, total cell RNA was extracted from THP-1 cells and p47-phox genes, which are the most highly regulated compo- by the guanidine HCl method (65) and analyzed by Northern blots per- by guest on September 27, 2021 formed according to standard procedures (66) or slot blots prepared ac- nents of the NADPH oxidase system and the most frequent sites cording to the instructions of Schleicher & Schuell (Keene, NH) for their for mutations that lead to chronic granulomatous disease (3). Minifold II apparatus. THP-1 cells were cultured with IFN-␥ (100 U/ml) and TNF-␣ (1000 U/ml) alone or in combination for up to 7 days in the presence of dexamethasone or indomethacin as described above. Hybrid- Materials and Methods ization probes were full-length cDNAs for human gp91-phox (67) and Cell culture conditions and experimental protocol p47-phox (18). Procedures for sequential cycles of prehybridization, THP-1 cells (57, 58) were cultured in RPMI 1640 complete medium sup- washes, and filter stripping were performed as described by Gatti et al. (68). Equal loading of lanes was demonstrated by examination of gels after plemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 ␮g/ml streptomycin, and 50 ␮M 2-ME at 37°C in a humid- ethidium bromide staining and by rehybridization with a 5.8-kb HindIII ␥ ␣ restriction fragment of rat 18S ribosomal cDNA (69). Positive control RNA ified 5% CO2 atmosphere with IFN- (100 U/ml) and TNF- (1000 U/ml), alone or in combination, for up to 7 days. All reagents were endotoxin free was obtained from differentiated HL-60 cells, and negative control RNA (Ͻ10 pg/ml as tested by Limulus amebocyte lysate assay). As indicated, was obtained from HeLa cells (26, 70). THP-1 cells received either dexamethasone (0.1–1.0 ␮M) or indomethacin Transcriptional regulation of gp91-phox and p47-phox was assessed by ␮ nuclear run-on transcription assays with minor modifications of previously in a conventional cyclo-oxygenase blocking concentration (10–20 M) ␥ (45–47) 1 day before starting the cytokine-induced differentiation process published procedures (71). THP-1 cells were cultured with IFN- (100 U/ml) plus TNF-␣ (1000 U/ml) for 2 days in the presence or the absence to allow intracellular accumulation and functional activity before cyto- ␮ kine stimulation (38–41, 48, 49, 59, 60). We chose dexamethasone as of 1 M dexamethasone. Based on the changes in expression of both gp91- phox and p47-phox transcripts in THP-1 cells cultured with IFN-␥ (100 a standard laboratory representative of the glucocorticoid agents and ␣ indomethacin as an archetypal example of a NSAID. Indomethacin was U/ml) and TNF- (1000 U/ml) for 2 days, we applied these conditions to preferred over other NSAIDs because it is a very well-characterized the run-on assays. Briefly, THP-1 nuclei were isolated by cell lysis in nonspecific cyclo-oxygenase inhibitor, besides being widely used in 0.05% Nonidet P-40. Freshly prepared nuclei were incubated for 30 min at 32 ␮ clinical medicine (61, 62). Cell counts and viability, monitored on a 30°C in a reaction mixture containing [ P]UTP (250 Ci, 3000 Ci/mmol) daily basis, were always Ͼ80%. 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 Superoxide release HCl method (65). Equal amounts of incorporated label from each group (1–2 ϫ 107 cpm) were then hybridized to saturating amounts of cDNA Superoxide release was assessed by a modified superoxide dismutase probes, immobilized on filters by slot blotting. The probes used in these (SOD) inhibitable cytochrome c reduction assay (63). Briefly, THP-1 cells experiments included cDNAs for the genes gp91-phox (67) and p47-phox were cultured in six-well polystyrene plates (1 ϫ 106 cells/well) with (18), a hybridization negative control (plasmid without insert), and a con- IFN-␥ (100 U/ml) and TNF-␣ (1000 U/ml), alone or in combination, for up stitutively expressed gene (␤-actin or ␣-tubulin) (72). to 7 days in the presence of dexamethasone or indomethacin as described Hybridization levels in Northern blots and nuclear run-on assays were above. On the day of the experiment (day 2 or 7), the plates were centri- quantified by PhosphorImager (Molecular Dynamics, Sunnyvale, CA) and fuged, the supernatant was removed, and the cytokine-differentiated THP-1 analyzed by ImageQuant software (Molecular Dynamics). Hybridization cells were incubated in HBSS (without phenol red) containing cytochrome levels in nuclear run-on assays were normalized to a hybridization negative c (50 ␮M) and the required cytokines for1hat37°C in a humidified 5% control (plasmid alone) and to constitutively expressed genes (␤-actin or ␣ CO2 atmosphere. Half the wells received SOD (60 U/ml) at the beginning -tubulin) and were calculated as relative rates of transcription (27). 4962 GLUCOCORTICOIDS INHIBIT NADPH OXIDASE GENE EXPRESSION

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 Ͻ 0.05 was considered signiﬁcant. Results 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-␥ (100 U/ml) and TNF-␣ (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 cul- Downloaded from tured under basal conditions (Fig. 1; p Ͻ 0.05 in all situations; n ϭ 6). Noteworthy is the tremendous synergism between IFN-␥ and TNF-␣. We simultaneously investigated the effect of dexamethasone (0.1, 0.5, or 1.0 ␮M) on the NADPH oxidase activity of THP-1

cells differentiated with IFN-␥ (100 U/ml) and TNF-␣ (1000 U/ml) http://www.jimmunol.org/ alone or in combination during 2 or 7 days. Figure 1 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 ␮M inhibited PMA-stimulated superoxide release by THP-1 cells on each day examined regardless of the cytokine combination ( p Ͻ 0.05 in all situations; n ϭ 6). At a 0.5-␮M concentration and on the second day of cell culture (Fig. 1A), dexamethasone inhib- by guest on September 27, 2021 ited PMA-stimulated superoxide release only of TNF-␣-differen- FIGURE 1. Dexamethasone inhibits the NADPH oxidase activity of cy- tiated THP-1 cells ( p Ͻ 0.05; n ϭ 6); however, on the seventh day tokine-differentiated THP-1 cells. THP-1 cells differentiated with IFN-␥ ␣ of cell culture (Fig. 1B), 0.5 ␮M dexamethasone inhibited PMA- (100 U/ml) and TNF- (1000 U/ml) alone or in combination for 2 days (A) or 7 days (B) show a higher PMA (30 nM)-stimulated superoxide release stimulated superoxide release by THP-1 cells cultured with any of Ͻ ϭ ␥ Ͻ ϭ (#, p 0.05; n 6). Note the strong synergism between IFN- (100 U/ml) the cytokines ( p 0.05 in all situations; n 6). Dexamethasone and TNF-␣ (1000 U/ml; A and B). Dexamethasone (Dexa) inhibits PMA- ␮ ,ء) at a 0.1- M concentration inhibited PMA-stimulated superoxide stimulated superoxide release of cytokine-differentiated THP-1 cells release only of THP-1 cells cultured with IFN-␥ and TNF-␣ for 7 p Ͻ 0.05; n ϭ 6; A and B) depending on the day of cell culture, cytokine days (Fig. 1B; p Ͻ 0.05; n ϭ 6). At other points, inhibition of combination, and dexamethasone concentration (0.1, 0.5, or 1.0 ␮M). Re- NADPH oxidase activity by dexamethasone was observed, but did sults are represented by boxplots showing the minimum, 25th percentile, not reach statistical signiﬁcance ( p Ͼ 0.05; n ϭ 6). median, 75th percentile, and maximum values.

We further assessed the cytochrome b558 content of THP-1 cells differentiated with IFN-␥ (100 U/ml) and TNF-␣ (1000 U/ml) alone or in combination for 2 or 7 days. Figure 2 shows that THP-1 U/ml) alone caused median 5-fold (day 2) and 13-fold (day 7) cells differentiated with these cytokines have a higher cytochrome increases in gp91-phox gene expression and 5-fold (day 2) and ␥ b558 content on both days 2 and 7 of cell culture than THP-1 cells 4-fold (day 7) increases in p47-phox gene expression. IFN- (100 cultured under basal conditions ( p Ͻ 0.05 in all situations; n ϭ 6). U/ml) combined with TNF-␣ (1000 U/ml) caused median 18-fold Again, a significant synergism between IFN-␥ and TNF-␣ oc- (day 2) and 51-fold (day 7) increases in gp91-phox gene expres- curred. Dexamethasone (1 ␮M) caused a statistically significant sion, and 12-fold (day 2) and 20-fold (day 7) increases in p47-phox ␣ reduction in cytochrome b558 content only in THP-1 cells cultured gene expression. TNF- (1000 U/ml) alone caused median 1.5- with IFN-␥ (100 U/ml) and TNF-␣ (1000 U/ml) for 7 days ( p Ͻ fold (day 2) and 3-fold (day 7) increases in gp91-phox gene ex- 0.05; n ϭ 3; Fig. 2B). At other points, dexamethasone caused a pression and 1.5-fold (day 2) and 2.8-fold (day 7) increases in Ͻ ϭ reduction of the cytochrome b558 content of cytokine-differentiated p47-phox gene expression ( p 0.05 in all situations; n 3; cal- THP-1 cells. This effect, however, was not statistically significant culations based on relative gene expression assessed by computer ( p Ͼ 0.05; n ϭ 3). analysis of PhosphorImager data). Figure 3 also shows the inhib- Considering the results showing the inhibitory effects of dexa- itory effect of dexamethasone (1 ␮M) on the expression of gp91- methasone on the NADPH oxidase activity of THP-1 cells differ- phox and p47-phox genes in THP-1 cells differentiated with IFN-␥ entiated with cytokines, we extended our investigation to the gene (100 U/ml) and TNF-␣ (1000 U/ml) alone or in combination dur- expression level as assessed by Northern blot hybridization (66). ing 2 or 7 days ( p Ͻ 0.05; n ϭ 3; calculations based on relative Figure 3 shows the induction of gp91-phox and p47-phox gene gene expression assessed by computer analysis of PhosphorImager expression in cytokine-differentiated THP-1 cells. IFN-␥ (100 data). It is noteworthy that dexamethasone inhibited approximately The Journal of Immunology 4963 Downloaded from

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-␥ (100 U/ml) and

TNF-␣ (1000 U/ml) alone or in combination induce the expression of http://www.jimmunol.org/ 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-␥ and TNF-␣. Dexamethasone (Dexa; 1 ␮M) 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 Ͻ 0.05 in all situations; n ϭ 3; calculations based on relative gene expression FIGURE 2. The effect of dexamethasone on the cytochrome b con- 558 assessed by computer analysis of PhosphorImager data. C, control; I, tent of cytokine-differentiated THP-1 cells. THP-1 cells differentiated with IFN-␥; T, TNF-␣. IFN-␥ (100 U/ml) and TNF-␣ (1000 U/ml) alone or in combination for 2

Ͻ by guest on September 27, 2021 days (A) or 7 days (B) have a higher cytochrome b558 content (#, p 0.05; n ϭ 6). Again, a strong synergism between IFN-␥ (100 U/ml) and TNF-␣ (1000 U/ml) occurs (A and B). Dexamethasone (Dexa; 1 ␮M) causes a signiﬁcant reduction of the cytochrome b558 content in THP-1 cells cul- for the gene expression studies as assessed by Northern blot p Ͻ hybridization. Transcription of both genes showed median 50% ,ء) tured with IFN-␥ (100 U/ml) and TNF-␣ (1000 U/ml) for 7 days ϭ 0.05; n 3; B). Results are represented by boxplots showing the minimum, inhibition ( p Ͻ 0.05; n ϭ 3). 25th percentile, median, 75th percentile, and maximum values. Effect of indomethacin on the NADPH oxidase activity,

cytochrome b558 content, and gene expression of gp91-phox and 50% of gp91-phox and p47-phox gene expression in THP-1 cells p47-phox in cytokine-differentiated THP-1 cells regardless of the cytokine treatment. Furthermore, in the absence Indomethacin (10 or 20 ␮M) showed a weak inhibitory effect on of dexamethasone, gp91-phox and p47-phox gene expression in the NADPH oxidase activity of cytokine-differentiated THP-1 THP-1 cells differentiated with cytokines for 2 or 7 days correlated cells (Table I). The drug failed to inhibit PMA-stimulated super- with their NADPH oxidase activity and cytochrome b content. oxide release by THP-1 cells cultured with cytokines for 2 days We further investigated the effect of dexamethasone (1 ␮M) on ( p Ͼ 0.05 in all situations; n ϭ 5). However, on the seventh day the transcription rates of gp91-phox and p47-phox genes in nuclei of cell culture, indomethacin (10 or 20 ␮M) significantly inhibited obtained from cytokine-differentiated THP-1 cells, as assessed by PMA-stimulated superoxide release by THP-1 cells cultured with nuclear run-on assays (71). Based on changes in the expression of IFN-␥ (100 U/ml) and TNF-␣ (1000 U/ml; p Ͻ 0.05; n ϭ 5). both gp91-phox and p47-phox transcripts in THP-1 cells cultured Indomethacin (10 ␮M) did not have any inhibitory effect on the with IFN-␥ (100 U/ml) and TNF-␣ (1000 U/ml) for 2 days, we cytochrome b content of cytokine-differentiated THP-1 cells applied the run-on assays to these conditions. As shown in Figure 558 (Table I) regardless of the cytokine treatment or the day of cell 4, the results demonstrate increased transcription rates of the genes culture ( p Ͼ 0.05 in all situations; n ϭ 4). Similarly, indomethacin encoding gp91-phox and p47-phox in THP-1 cells cultured with (10 ␮M) failed to inhibit the expression of gp91-phox and p47- IFN-␥ (100 U/ml) and TNF-␣ (1000 U/ml) for 2 days in parallel phox genes in cytokine-differentiated THP-1 cells on day 2 or 7 of with Northern blot experiments. Transcription rates showed, re- cell culture ( p Ͼ 0.05; n ϭ 3; results not shown). spectively, 5- and 7.5-fold increases ( p Ͻ 0.05; n ϭ 3; calculations of relative transcription rates normalized to negative control and to rates for the constitutively expressed genes ␣-tubulin and ␤-actin, Discussion assessed by computer analysis of PhosphorImager data). Dexa- Macrophages play a central role in inflammation (1, 2). The oxy- methasone (1 ␮M) significantly inhibited the transcription rates gen-derived metabolites generated by the phagocyte NADPH ox- of gp91-phox and p47-phox genes in THP-1 cells cultured with idase provide an important defense mechanism, but also present a IFN-␥ and TNF-␣ alone, confirming our previous observations risk for tissue damage (5, 6). Cytokines and anti-inflammatory 4964 GLUCOCORTICOIDS INHIBIT NADPH OXIDASE GENE EXPRESSION Downloaded from

FIGURE 4. Dexamethasone inhibits the transcription of gp91-phox and p47-phox genes in THP-1 cells differentiated with IFN-␥ (100 U/ml) and TNF-␣

(1000 U/ml). A representative nuclear run-on experiment is presented showing the inhibitory effect of dexamethasone (Dexa; 1 ␮M) on the transcription http://www.jimmunol.org/ rates of gp91-phox and p47-phox genes in THP-1 cells differentiated with IFN-␥ (100 U/ml) and TNF-␣ (1000 U/ml) for 2 days (p Ͻ 0.05; n ϭ 3; calculations of relative transcription rates normalized to the negative control and to rates for the constitutively expressed genes ␣-tubulin and ␤-actin, assessed by computer analysis of PhosphorImager data).

drugs such as glucocorticoids and NSAIDs are important regula- b558 content increased, respectively, 2-, 1.5-, and 5-fold. Expression tors of inﬂammation (76) at least in part due to their effects on of the gene encoding the cytochrome b558 component gp91-phox in- phagocyte oxidant production (30–37, 50–53). creased, respectively, 5-, 1.5-, and 18-fold on day 2 and 13-, 3-, and ␥ Human monocytic THP-1 cells have well-characterized IFN- and 51-fold on day 7. Expression of the gene encoding the cytosolic ox- by guest on September 27, 2021 TNF-␣ receptors (77, 78). Our results show that IFN-␥ and TNF-␣ idase component p47-phox increased, respectively, 5-, 1.5-, and 12- alone or in combination induce myelomonocytic THP-1 cells to dif- fold on day 2 and 4-, 2.8-, and 20-fold on day 7. Nuclear run-on ferentiate and express NADPH oxidase activity, cytochrome b558, and assays showed respective 5- and 7.5-fold increases in gp91-phox and gene transcripts for the NADPH oxidase components gp91-phox and p47-phox gene transcription on the second day of THP-1 cell culture p47-phox. Incubation with IFN-␥, TNF-␣, or the highly synergistic with IFN-␥ plus TNF-␣. combination induced, respectively, 10-, 2-, and 100-fold increases in Despite the widespread use of glucocorticoids, the molecular PMA-stimulated superoxide release by THP-1 cells. The cytochrome mechanisms that underlie their therapeutic effects are poorly

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

Control IFN-␥ TNF-␣ IFN-␥ ϩ TNF-␣

Superoxide releasec Day2(n ϭ 5) Control 0.09 (0.08–0.13) 1.89 (1.72–1.93) 0.78 (0.74–0.87) 51.31 (48.54–54.82) Indomethacin (10 ␮M) 0.09 (0.08–0.12) 2.21 (2.00–2.27) 0.72 (0.70–1.00) 56.92 (56.28–59.08) Indomethacin (20 ␮M) 0.03 (0.03–0.05) 2.49 (2.48–2.82) 0.91 (0.89–0.97) 47.98 (45.63–48.45) Day7(n ϭ 5) Control 0.13 (0.11–0.14) 2.31 (2.14–2.66) 0.87 (0.86–0.94) 69.71 (67.33–70.83) Indomethacin (10 ␮M) 0.14 (0.14–0.15) 2.45 (1.93–2.62) 0.98 (0.94–1.01) 58.91 (58.20–60.24)* Indomethacin (20 ␮M) 0.09 (0.09–0.12) 2.13 (1.96–2.33) 0.94 (0.89–0.95) 57.23 (54.01–58.18)* b Cytochrome b558 content Day2(n ϭ 4) Control 11.29 (10.54–11.79) 17.95 (16.12–20.28) 16.23 (16.12–16.94) 39.02 (37.77–39.50) Indomethacin (10 ␮M) 11.21 (10.61–11.32) 14.94 (13.55–15.33) 15.46 (14.62–15.78) 41.94 (37.62–42.13) Day7(n ϭ 4) Control 10.91 (9.86–11.71) 19.23 (17.12–20.40) 13.97 (13.50–15.60) 40.53 (38.84–40.89) Indomethacin (10 ␮M) 10.02 (9.51–12.00) 17.43 (16.24–17.46) 17.15 (15.16–17.74) 42.22 (39.96–42.28)

a Indomethacin (10 or 20 ␮M) inhibits PMA-stimulated superoxide release by THP-1 cells differentiated with IFN-␥ (100 U/ml) and TNF-␣ (1000 U/ml) for 7 days (* p Ͻ ϭ ␮ ␥ ␣ 0.05, n 5). Indomethacin (10 M) did not inhibit the cytochrome b558 content of THP-1 cells differentiated with IFN- (100 U/ml), TNF- (1000 U/ml) alone, or in combination for 2 or 7 days (p Ͼ 0.05, n ϭ 4). b Effect of indomethacin on the PMA (30 nM) stimulated superoxide release by cytokine-differentiated THP-1 cells in nmol superoxide/106 cells/h: median (25th–75th percentile). c Effect of indomethacin on the cytochrome b558 content of cytokine-differentiated THP-1 cells in pmol cytochrome b/mg of total protein median. The Journal of Immunology 4965

understood. Among several effects, glucocorticoids are known (54), and in phagocytes from patients with variant forms of chronic to inhibit the production and gene expression of many cyto- granulomatous disease, small changes in gp91-phox content in- kines, including IL-1␣, IL-1␤, IL-2, IL-3, IL-4, IL-5, IL-6, duced by IFN-␥ produce seemingly disproportionate increases in IL-8, IL-10, TNF-␣, TNF-␤, IFN-␥, and growth macrophage respiratory burst activity (55, 56). CSF (79–88). Glucocorticoids interact with transcription fac- IFN-␥ was a stronger stimulus than TNF-␣ for the induction of ␬ tors, including activating protein-1 (38–40) and NF- B (41), to oxidase gene expression, cytochrome b558 content and superoxide down-regulate gene expression. Our studies indicate that dexa- release. The induction of gp91-phox gene expression in THP-1 methasone down-regulated the NADPH oxidase system at least cells by IFN-␥ alone was previously reported by our group (26). in part by inhibiting the transcription of gp91-phox and p47- THP-1 cells were less responsive to TNF-␣ than human peripheral phox genes. The molecular mechanisms involved in this pro- blood monocytes/macrophages (27). The up-regulation of gp91- cess, such as potential interaction of dexamethasone with tran- phox correlated qualitatively, but not quantitatively, with p47- scription factors, are under current investigation in our phox. The gp91-phox gene expression increased in a more dra- laboratories. matic way than that of p47-phox. Whether the gp91-phox gene is The production of endogenous inhibitors regulating the NADPH more important than the p47-phox gene for the up-regulation of the oxidase system and the inhibition of the protein kinase C (PKC) NADPH oxidase system remains to be determined. Indeed, pathway by dexamethasone are likely events in our model system X-linked chronic granulomatous disease has generally a more se- that could partially explain the inhibition of PMA-stimulated su- vere clinical course than the autosomal form of the disease, which peroxide release by cytokine-differentiated THP-1 cells. Depend- is commonly due to defects in p47-phox (101–103). ing on the cell lineage and experimental conditions, glucocorti- Our studies also demonstrate that indomethacin (10 or 20 ␮M) Downloaded from coids either inhibit (89, 90) or do not affect the PKC pathway (91, inhibited PMA-stimulated superoxide release by THP cells differ- 92). We have focused our investigation on gp91-phox and p47- entiated with IFN-␥ and TNF-␣ for 7 days, but not to the extent phox gene expression studies and correlated them with cytochrome caused by dexamethasone. This effect may be attributed in part to

b558 content and NADPH oxidase activity. We propose the regu- the indomethacin inhibition of both cyclo-oxygenase isoforms (61, lation of NADPH oxidase gene expression by glucocorticoids as 62). At lower levels of PMA-stimulated superoxide release by

an additional new mechanism for the effect of glucocorticoids on THP-1 cells differentiated with IFN-␥ or TNF-␣ alone, the indo- http://www.jimmunol.org/ phagocyte oxidase activity. Investigation of other endogenous in- methacin inhibitory effect on NADPH oxidase activity could not hibitors or speciﬁc inhibition of the PKC pathway in our model be detected. In addition, indomethacin did not affect the cells’ cy-

system constitutes a major subject for future investigation. tochrome b558 content or expression of the genes encoding gp91- In contrast, studies in both THP-1 cells and other systems have phox and p47-phox. A direct quenching of active oxygen species shown that glucocorticoids can also enhance cytokine responses. by indomethacin (104), the influence of cyclo-oxygenase isoforms For example, dexamethasone and IL-1 synergize to stimulate the in our model system, or other interfering cellular mechanisms re- production of granulocyte CSF in differentiated THP-1 cells (93). main to be determined. The in vitro enhancement of superoxide anion release stimulated We conclude that dexamethasone inhibited the NADPH oxidase by Mycobacterium leprae or Mycobacterium bovis shows re- activity of cytokine-differentiated THP-1 cells at least in part by by guest on September 27, 2021 sponses to lower concentrations of IFN-␥ in monocytes from lep- down-regulating the transcriptional expression of genes encoding rosy patients receiving prednisone therapy than in monocytes from components of the NADPH oxidase system. Indomethacin inhib- healthy subjects or from other leprosy patients (94). TNF-␣ in- ited only the NADPH oxidase activity of cytokine-differentiated creases glucocorticoid-induced transcriptional activity of the glu- THP-1 cells. This mechanism of action of glucocorticoids may be cocorticoid receptor via the glucocorticoid response elements in clinically relevant to patients suffering from inflammatory diseases mouse fibroblasts (95). These phenomena may reflect a general due to the harmful effects of the excessive release of oxygen-de- molecular mechanism by which cytokines or glucocorticoids mod- rived metabolites. ulate the transcriptional activity of their receptors, providing counter-regulatory mechanisms at the level of their target cells. References All measures demonstrated tremendous synergism between IFN-␥ and TNF-␣ induction of NADPH oxidase activity and ex- 1. Henderson, L. M., and J. B. Chappell. 1996. NADPH oxidase of neutrophils. Biochim. Biophys. Acta 1273:87. pression of its components. This synergism has also been observed 2. Chanock, S. J., J. El Benna, R. M. Smith, and B. M. Babior. 1994. The respi- in a different model system (96). The molecular mechanisms for ratory burst oxidase. J. Biol. Chem. 269:24519. the synergy between IFN-␥ and TNF-␣ remain to be investigated. 3. Curnutte, J. T., S. H. Orkin, and M. C. Dinauer. 1994. Genetic disorders of phagocyte function. In The Molecular Basis of Blood Diseases, 2nd Ed. The nuclear run-on assays indicate that the mechanism of up- G. Stamatoyannopoulos, A. W. Neinhuis, P. W. Majerus, and H. Varmus, eds. regulation of the NADPH oxidase system in our model is at least W. B. Saunders, Philadelphia, p. 493. 4. Quie, P. G. 1993. Chronic granulomatous disease of childhood: a saga of dis- in part transcriptional. The even larger change in steady state levels covery and understanding. Pediatr. Infect. Dis. J. 12:395. of the gene transcripts could represent either accumulation of 5. McCord, J. M., B. Gao, J. Leff, and S. C. Flores. 1994. Neutrophil-generated mRNA due to higher levels of production than degradation or ac- free radicals: possible mechanisms of injury in adult respiratory distress syn- drome. Envirn. Health Perspect. 102(Suppl. 10):57. tive regulation of mRNA stability. Other post-transcriptional 6. Halliwell, B. 1995. Oxygen radicals, nitric oxide and human inflammatory joint mechanisms, such as translational enhancement, calcium mobili- disease. Ann. Rheum. Dis. 54:505. zation (97), protein phosphorylation (98), G protein activation 7. Wiseman, H., and B. Halliwell. 1996. Damage to DNA by reactive oxygen and nitrogen species: role in inflammatory disease and progression to cancer. Bio- (99), or phospholipase A2 activation (100), could contribute to the chem. J. 313:17. activation of the NADPH oxidase system and hence the quantita- 8. Segal, A. W., O. T. G. Jones, D. Webster, and A. C. Allison. 1978. Absence of a newly described cytochrome b from neutrophils of patients with chronic gran- tive differences between the cytochrome b558 content and the ulomatous disease. Lancet ii:446. NADPH oxidase activity results. 9. Segal, A. W., and A. Abo. 1993. The biochemical basis of the NADPH oxidase As might be expected from a multicomponent enzyme system, of phagocytes. Trends Biochem. Sci. 18:43. NADPH oxidase activity does not strictly correlate with the 10. Jesaitis, A. J., E. S. Buescher, D. Harrison, M. T. Quinn, C. A. Parkos, S. Livesey, and J. Linner. 1990. Ultrastructural localization of cytochrome b in amount of any single component. For example, eosinophils sup- the membranes of resting and phagocytosing human granulocytes. J. Clin. In- port considerable oxidase activity with low levels of gp91-phox vest. 85:821. 4966 GLUCOCORTICOIDS INHIBIT NADPH OXIDASE GENE EXPRESSION

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