Nitric Oxide Is an Important Regulator of Heme Oxygenase-1 Expression in the Lipopolysaccharide and Interferon-γ-Treated Murine Macrophage-Like Cell Line J774.1/JA-4

Abstract

Heme oxygenase-1 (HO-1) catabolizes the degradation of heme into bilirubin, carbon monoxide, and iron ions. The HO-1 products provide antioxidant cytoprotection in addition to having potent antiinflammatory and immunomodulatory functions. HO-1 is induced by its substrate heme and environmental factors including oxidative and heat stresses. Although previous studies reported that lipopolysaccharide (LPS) induced the expression of both the HO-1 gene and its protein in macrophages, the major regulators of HO-1 expression remain unknown. To identify these regulators, we used two types of cell, the murine macrophage-like cell line J774.1/JA-4 and its LPS-resistant mutant, LPS1916. Based on a comparison of the results obtained with these cells, we found that nitric oxide (NO) was closely linked to the induction of HO-1. Real-time polymerase chain reaction (PCR) showed that the time course for inducible HO-1 mRNA by LPS or LPS+interferon (IFN)-γ was similar to that for inducible NO synthase (iNOS) mRNA. Furthermore, the expression of iNOS mRNA and protein increased earlier than that of HO-1 mRNA and protein. N-Nitro-L-arginine methyl ester, an NO synthase inhibitor, reduced both HO-1 expression and NO production in LPS+IFN-γ-treated JA-4 cells. Furthermore, NOC-12, an NO donor, significantly induced HO-1 expression not only in JA-4 but also in LPS1916 cells. Reactive oxygen species (ROS) scavengers, such as superoxide dismutase and catalase, did not affect HO-1 protein expression in LPS+IFN-γ-treated JA-4 cells. These results suggest that, among ROS, NO plays an important role in HO-1 induction in activated macrophages treated with LPS+IFN-γ.

Macrophages reside in almost all tissues and are involved as important effector cells at all stages of innate and adaptive immune responses. Macrophages are vigorously involved in host defense against a myriad of potentially pathogenic infectious agents in the outside environment.^1,2) Macrophages use a wide variety of pattern recognition receptors including Toll-like receptors (TLRs) that bind to these agents.^3,4) Such receptors recognize various microbial components.⁵⁾ Once TLRs sense the presence of invading pathogens, their engagement with them activates macrophages, which then produce and release various sets of effector molecules aimed at destroying the foreign agents. Reactive oxygen species (ROS), such as superoxide anion (O₂⁻), hydrogen peroxide (H₂O₂), and nitric oxide (NO), are their front line effector molecules.^6–11) These highly diffusive products exert strong cytotoxic activities against micro-organisms and many neighboring cells, including against the macrophages themselves. However, macrophages and most of the aerobic host cells can protect themselves by enhancing the expression of heme oxygenase (HO). HO, a cellular antioxidant, is one of the key enzymes catalyzing the degradation of heme-containing molecules to biliverdin, free iron, and carbon monoxide.^12,13) Three HO isozymes, including HO-1 (HSP32), HO-2, and HO-3, have been identified¹⁴⁾; although HO-3 may be a pseudo gene derived from HO-2 transcripts.¹⁵⁾ HO-1 is an inducible enzyme, whereas HO-2 is constitutive one.¹⁶⁾ HO-1 is induced by various molecules and environmental factors, such as heme, radiation, cytokine, oxidative, and heat stresses.^17–21) Previous studies reported that lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, also induces the expression of both the HO-1 gene and its protein in macrophages.^22–24) Exposure of HO-1-deficient mice to LPS leads to increased hepatocellular necrosis, increased splenic proinflammatory cytokine secretion, and higher mortality of septic shock when compared with wild-type animals.^25,26) These studies suggest that HO-1 plays an important role in reducing the deleterious increase in oxidative damage. Srisook et al. reported that both O₂⁻ and NO are related to HO-1 induction in macrophages by using an O₂⁻ generator and a NO donor.²⁷⁾ However, it is not easy to study the role of O₂⁻ and NO separately in HO-1 induction in activated macrophages which produce both of these molecules simultaneously after LPS-treatment. Thus, among ROS, the important regulators of HO-1 in the activated macrophages have not been clear. The aim of the present work was to identify the regulator of HO-1 induction in activated macrophages. In this study, we sought to identify the regulator of HO-1 induction of the activated macrophages by using 2 types of macrophages, i.e., cells of the murine macrophage-like cell line J774.1/JA-4 and their LPS-resistant mutant, LPS1916.⁷⁾ This mutant cell line is characterized by a specific defect in the induction of ROS after LPS treatment. Our recent study reported that LPS1916 cells have the phenotype of mal-expression of CD14, a key molecule required for LPS-induced activation of cells, on the macrophage cell surface, leading to reduced responses to LPS.²⁸⁾ Interestingly, this defect in ROS induction in LPS-treated LPS1916 cells is partly overcome by the simultaneous addition of interferon (IFN)-γ.²⁹⁾ Therefore, we expected to reveal the role of O₂⁻ and NO for HO-1 expression by macrophage mutant cells. We treated JA-4 and LPS1916 cells with LPS and/or IFN-γ, and we explored the correlation between the expression of HO-1 and the generation of ROS. We found that NO played an important role in HO-1 induction in the activated macrophages treated with LPS+IFN-γ.

MATERIALS AND METHODS

Materials

Escherichia coli 055:B5 LPS, chromatographically purified, was obtained from Sigma-Aldrich (St. Louis, MO, U.S.A.). Ham’s F12 medium was purchased from Life Technologies (Carlsbad, CA, U.S.A.); and fetal bovine serum (FBS), containing less than 60 pg LPS per mL, from Life Technologies. Recombinant murine IFN-γ was a generous gift from TORAY (Tokyo, Japan). Penicillin and streptomycin solution was purchased from Nacalai Tesque (Kyoto, Japan). Phorbol myristate acetate (PMA), cytochrome c from horse heart, superoxide dismutase (SOD) from bovine liver (≥1500 units/mg protein) and catalase from bovine liver (2000–5000 units/mg protein) were obtained from Sigma-Aldrich. Apocynin was purchased from Cayman Chemical Company (Ann Arbor, MI, U.S.A.). All other reagents and chemicals were of the purest commercial grade available.

Cell Culture

Culturing of the JA-4 cell line, an LPS-sensitive subline of the murine macrophage-like cell line J774.1, and that of the LPS1916 cell line, an LPS-resistant mutant cell line originating from JA-4,⁷⁾ were performed as described previously.³⁰⁾ In brief, the cells were maintained and cultured in 10 mL of Ham’s F-12 medium supplemented with 10% heat-inactivated FBS, penicillin and streptomycin (50 units/mL and 50 µg/mL, respectively) in 100-mm plastic dishes (Falcon #351029, Corning Life Science, NY, U.S.A.) at 37°C in a CO₂ incubator (5% CO₂/95% humidified air).

Assay for O₂⁻-Generating Activity and NO₂⁻ Production

O₂⁻ generation activity was examined as described before.³¹⁾ Cells were seeded at 1×10⁵ cells per well into 48-well plates (Costar #3548, Corning Life Science) and then incubated at 37°C for 17 h. The medium was replaced with fresh medium containing LPS (100 ng/mL) and/or IFN-γ (10 units/mL), and the cells were incubated at 37°C for 20 h. Then, the culture media were collected and stored at −30°C until analyzed. The cells were washed twice with phosphate buffered saline (PBS) and then covered with 0.25 mL Hank’s balanced salt solution containing CaCl₂ (1 mM), MgCl₂ (1 mM), and cytochrome c (0.625 mg/mL) with or without SOD (0.03 mg/well). The reaction was initiated by the addition of PMA (5 µg/mL), followed by incubation at 37°C for 90 min, and then stopped by chilling the cells on ice. The supernatants were examined at a wavelength of 550 nm by a UV-160 photometer (Shimadzu, Kyoto, Japan). The differences in A₅₅₀ between the samples without and with SOD were determined, and the amounts of O₂⁻ generated were calculated as the reduction of cytochrome c on the basis of the fact that 1 unit of optical density at 550 nm corresponds to 47.2 nmol of O₂⁻. The results were expressed as specific activities divided by the amounts of cellular proteins in wells recovered after washing off the reaction mixture and quantitated by the method of Lowry et al.³²⁾ Nitric oxide (NO) was measured as a stable form of nitric anion (NO₂⁻) by using Griess reagent (Wako Pure Chemical Industries, Ltd., Osaka, Japan). A total of 100 µL of supernatant was combined with an equal volume of Greiss reagent (6 mg/mL), and the samples were incubated at room temperature for 5 min. The reaction products were calorimetrically quantified at 550 nm with background subtraction at 630 nm, using a Multiscan plus microplate reader (Labsystems Multiskan MS, Dainippon Sumitomo Pharma Co., Ltd., Osaka, Japan). The nitrite concentration was calculated with reference to a sodium nitrite standard.

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)/Western Blotting

As described previously, the cells were seeded at 2×10⁶ cells/5 mL/dish (Corning #430566) and incubated at 37°C for 17 h. Then, the cells were stimulated or not with LPS, LPS+IFN-γ or NOC12 (Dojindo Chemical, Kumamoto, Japan) for various times. The cells were then chilled on ice and washed twice with ice-cold PBS, after which they were scrapped into lysis buffer containing 20 mM N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid–sodium hydroxide (HEPES–NaOH) buffer, pH 7.5, 1% (v/v) Triton X-100, 2 mM ethylenediaminetetraacetic acid (EDTA), 150 mM NaCl, 10% glycerol and 1% protease inhibitor cocktail (Nacalai Tesque, Kyoto, Japan). Finally, the cell lysates were centrifuged at 10000×g for 1 min at 4°C, and the resultant supernatant was used as the cell extract. For SDS-PAGE/Western blotting, 25 µg aliquots of the cell extracts were treated with SDS-sample buffer and then boiled at 100°C for 5 min. The samples were thereafter loaded onto a 5–20% gradient polyacrylamide gel (ATTO, Tokyo, Japan), electrophoresed in a discontinuous buffer system of Laemli,³³⁾ and then electro-transferred to an Immobilon polyvinylidene difluoride (PVDF) membrane (Merck, Millipore, Billerica, U.S.A.) at 30 V overnight and then at 100 V for 30 min at 4°C. After blocking the filter with 30 mg/mL milk casein (Megmilk Snow Brand, Tokyo, Japan) in a rinse buffer comprising 0.1% Triton X-100, 0.1 mM EDTA, and 0.8% NaCl in 10 mM Tris–HCl buffer, pH 7.5, the proteins on the filter were reacted with polyclonal HO-1 antibody (Enzo Life Sciences, NY, U.S.A.) or monoclonal β-actin antibody (Sigma-Aldrich) at room temperature for 2 h, and then the membrane filter was rinsed 3 times with the rinse buffer. Next, the membrane was reacted with horse-radish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse immunoglobulin G (IgG) (Cell Signaling Technology, Danvers, MA, U.S.A.) at room temperature for 1 h, and then the membrane filter was rinsed 3 times with the rinse buffer. The immune complexes on the membrane were detected by the addition of Pierce Western blotting Substrate (Thermo Fisher Scientific Inc., Waltham, MA, U.S.A.). Chemiluminescence signals were detected by using an LAS 3000 mini image analyzer (FUJIFILM, Tokyo, Japan), and the results were analyzed with Image J software.

RNA Isolation, Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) Analysis

Total RNAs were isolated from cells by using Tripure Isolation Reagent (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer’s instructions. RNA quality and concentration were assessed with a NanoDrop Light spectrophotometer (Thermo Fisher Scientific Inc.). Total RNAs were reverse-transcribed with a ReverTra Ace® qPCR RT Master Mix (TOYOBO, Osaka, Japan). Real-time PCR was performed on an applied Biosystems StepOnePlus™ Real-Time PCR System (Applied Biosystems; Life Technologies, Carlsbad, CA, U.S.A.) using FastStart Universal SYBR Green Master (ROX; Roche Diagnostics GmbH), and relative quantification (RQ) was calculated by using StepOne™ software V2.2.2, based on the equation RQ=2^−ΔΔCt, where Ct is the threshold cycle to detect fluorescence. Ct data were normalized to the internal standard GAPDH. Primer sets for qPCR-PCR are described in Table 1.

Table 1. Primer Used in qRT-PCR Expreriment

Name	Acc. No.	Forward primer	Reverse primer
HO-1	NM_010442.2	5′-AGG CTA AGA CCG CCT TCC T-3′	5′-TGT GTT CCT CTG TCA GCA TCA-3′
HO-2	NM_001136066.2	5′-GCG GAG ACT GAC TGA CCT G-3′	5′-TTG TGG CTG AGT AGT TTG TGC T-3′
Mn-SOD	NM_013671.3	5′-TGC TCT AAT CAG GAC CCA TTG-3′	5′-GTA GTA AGC GTG CTC CCA CAC-3′
Catalase	NM_009804.2	5′-CCT TCA AGT TGG TTA ATG CAG A-3′	5′-CAA GTT TTT GAT GCC CTG GT-3′
iNOS	NM_010927.3	5′-CTT TGC CAC GGA CGA GAC-3′	5′-TCA TTG TAC TCT GAG GGC TGA C-3′

Statistical Analysis

Results are expressed as the mean±S.E.M. of at least three experiments performed using in vitro cell preparations. Data for the two groups were analyzed using Student’s t-test, and data for more than two groups were compared using one-way ANOVA with Bonferroni multiple comparison as a post hoc test in Pharmaco basic software V14.2.2 (Three S, Tokyo, Japan). Statistical significance was set at p<0.05 or p<0.01.

RESULTS

Induction of HO-1 Protein in JA-4 and LPS1916 Cells Treated with LPS and/or IFN-γ

To examine the effect of LPS and/or IFN-γ on the induction of HO-1 in JA-4 and LPS1916 cells, we left the cells untreated or treated them with LPS (100 ng/mL), IFN-γ (10 units/mL) or both for 4 or 20 h, and then lysed them. The expression of HO-1 protein was determined by Western blot analysis. As shown in Fig. 1, HO-1 protein was induced in both cell lines in 4 h with or without treatment with LPS and/or IFN-γ. Moreover, when the medium was replaced with fresh medium and then incubated at 37°C, the high expression of HO-1 persisted for up to 8 h (data not shown), indicating that HO-1 had been induced by the medium change. After 20 h, however, the expression of HO-1 in untreated JA-4 and LPS1916 cells returned to the basal levels (Figs. 1A, B). In contrast, by this time LPS had slightly induced HO-1 protein expression, and LPS+IFN-γ significantly increased the expression in JA-4 cells (Fig. 1A); whereas HO-1 in LPS1916 cells was poorly induced by these treatments (Fig. 1B).

Fig. 1. Effect of LPS and/or IFN-γ on the Induction of HO-1 in JA-4 and LPS1916 Cells

JA-4 (A) or LPS1916 (B) cells were preincubated at 37°C for 17 h, and then the cells were treated with LPS (100 ng/mL), INF-γ (10 units/mL), or both for the indicated times. The cell lysates were prepared and subjected to Western blot analysis. The same filter membranes were probed again with an anti-β-actin antibody to ensure equal loading of cellular proteins onto the gel. The data were expressed relative to those for the untreated cells at 0 h after normalization with the amount of β-actin in each cell. Error bar represents the S.E.M. for 3 independent experiments. ** p<0.01 versus the untreated cells at 20 h.

Effect of LPS and/or IFN-γ on the O₂⁻ Generating Activity and NO Production in JA-4 and LPS1916 Cells

Various studies have shown that HO-1 is highly inducible by agents causing oxidative stress.^34,35) It is well known that activated macrophages produce ROS. Thus, to clarify the role of ROS in the induction of HO-1 in the activated macrophages, we determined O₂⁻ generating activity and NO production in JA-4 and LPS1916 cells. These cells were incubated with or without LPS and/or IFN-γ at 37°C. After 20 h of incubation, we determined the O₂⁻-generating activity by using the cytochrome c reduction assay and NO₂⁻ (a stable metabolite of NO) level by performing the Griess reagent assay. O₂⁻-generating activity was very low in the untreated JA-4 or LPS1916 cells (Figs. 2A, B). Incubation of JA-4 cells with LPS, IFN-γ, or LPS+IFN-γ enhanced the O₂⁻-generating activity; and among them, LPS alone and LPS+IFN-γ treatment markedly increased it (Fig. 2A). On the other hand, in LPS1916 cells, only one of these treatments, i.e., LPS+IFN-γ, markedly elevated the activity (Fig. 2B). As shown in Fig. 3A, although treatment with IFN-γ alone induced no NO₂⁻ accumulation in the culture medium of the JA-4 cells, the accumulation was induced by LPS treatment, and markedly so by LPS+IFN-γ treatment. The culture medium of the LPS1916 cells treated with LPS or IFN-γ alone showed no detectable NO₂⁻ accumulation, whereas there was moderate accumulation of NO₂⁻ when the cells were treated with LPS+IFN-γ (Fig. 3B).

Fig. 2. Induction of O₂⁻-Generating Activity by LPS, IFN-γ or Both

JA-4 and LPS1916 cells were preincubated at 37°C for 17 h, and then the cells were treated with LPS (100 ng/mL), INF-γ (10 units/mL) or both for 20 h. O₂⁻ generation was assayed in Hank’s balanced salt solution as SOD-sensitive cytochrome c reduction at 37°C for 90 min. The results are expressed as specific activities of the cells, normalized with cell proteins in the assay. Error bar represents the S.E.M. for 5 independent experiments performed in duplicate. * p<0.05; ** p<0.01 versus the untreated cells at 20 h.

Fig. 3. Induction of NO Production by LPS, IFN-γ or Both

JA-4 and LPS1916 cells were preincubated and then treated with LPS and/or IFN-γ as described in the legend to Fig. 2. NO₂⁻ in culture supernatants was quantified by performing the Griess reagent assay, as described in Materials and Methods. Error bar represents the S.E.M. for 5 independent experiments performed in duplicate. ** p<0.01 versus the untreated cells at 20 h.

Effects of LPS and LPS+IFN-γ on HO-1, HO-2, Mn-SOD, Catalase, and iNOS mRNA Expression Levels

As shown in Figs. 1–3, under the conditions of high O₂⁻-generating activity, macrophages treated with LPS (JA-4) or LPS+IFN-γ (LPS1916), no significant increase of HO-1 was observed. In contrast, under the conditions of high NO production, HO-1 was markedly induced in LPS+IFN-γ-treated JA-4 cells. So we hypothesized that NO but not O₂⁻ was involved in the induction of HO-1. To confirm the relationship between NO and HO-1 further, we next examined the expression patterns of genes encoding heme, O₂⁻, and H₂O₂ detoxification proteins (HO-1, HO-2, manganese superoxide dismutase [Mn-SOD], and catalase) and NO synthase (inducible NO synthase [iNOS]), in untreated, LPS-, and LPS+IFN-γ-treated JA-4 cells. Mn-SOD is one of 3 SODs; and it is induced by cytokines, LPS, and IFN-γ.^36–42) Among the 3 different isoforms of NOS, iNOS is the primary regulator of NO production in the innate immune system; and its expression can be induced by LPS, IFN-γ, IL-1β, IL-6, and TNF-α.⁴³⁾ JA-4 cells were incubated for various times at 37°C in the absence or presence of LPS or LPS+IFN-γ, and total RNA was then isolated from these cells at each time point. As shown in Fig. 4A, the apparent induction of HO-1 gene expression occurred at 4 h after LPS or LPS+IFN-γ treatment and continued to increase until 8 h, after which it remained relatively constant during the remainder of the time course. The expression level of the HO-1 gene achieved by treatment with LPS+IFN-γ was higher than that by treatment with LPS alone. Gene expression of HO-2 and catalase was not significantly influenced by treatment with LPS or LPS+IFN-γ (Figs. 4B, C). Mn-SOD mRNA was induced as early as 2 h after the start of LPS or LPS+IFN-γ treatment, reached its maximal expression level at 8 h, and then gradually decreased during the rest of the time course (Fig. 4D). The expression level of the Mn-SOD gene reached by treatment with LPS alone was similar to that by treatment with LPS+IFN-γ. iNOS gene expression was induced as early as 2 h after LPS or LPS+IFN-γ treatment, reached its maximum at 4 h, and then gradually decreased by 8 h. The expression level of the iNOS gene achieved by treatment with LPS+IFN-γ was much higher than that by treatment with LPS alone (Fig. 4E). These results show that the pattern of the time-course for HO-1 mRNA by LPS or LPS+IFN-γ was similar to that for iNOS mRNA, although the expression of the iNOS mRNA increased earlier than that of HO-1 mRNA.

Fig. 4. Induction of HO-1 and iNOS mRNAs in LPS or LPS+IFN-γ-Treated JA-4 Cells

JA-4 cells were preincubated for 17 h and then incubated without (△) or with LPS (100 ng/mL, □) or LPS (100 ng/mL) plus IFN-γ (10 units/mL, ○) for the indicated times. The expression levels of HO-1 (A), HO-2 (B), catalase (C), Mn-SOD (D), and iNOS (E) genes were determined by using quantitative RT-PCR, with the data normalized with the internal control GAPDH gene and the results expressed as values relative to those of the untreated cells at 0 h. Data are means and SEM for 3 independent experiments.

Effects of LPS and LPS+IFN-γ on HO-1 and iNOS Protein Expression Levels

To examine the relationship between HO-1 and iNOS further, we compared the expression of HO-1 and iNOS proteins in untreated, LPS- and LPS+IFN-γ-treated JA-4 cells. As shown in Fig. 5, iNOS protein expression was detected within 4 h after LPS+IFN-γ treatment, while HO-1 protein expression began to increase after 8 h. These results show a certain relationship between HO-1 and iNOS protein expression, as suggested by the expression of the corresponding gene (Figs. 4A, E).

Fig. 5. Induction of HO-1 and iNOS Protein in LPS or LPS+IFN-γ-Treated JA-4 Cells

JA-4 cells were preincubated for 17 h and then incubated without (△) or with LPS (100 ng/mL, □) or LPS (100 ng/mL) plus IFN-γ (10 units/mL, ○) for the indicated times. The cell lysates were prepared and subjected to Western blot analysis. The same filter membranes were probed again with an anti-β-actin antibody to ensure equal loading of cellular proteins onto the gel. The data were expressed relative to those for the untreated cells at 0 h after normalization with the amount of β-actin in each cell. Error bar represents the S.E.M. for 3 independent experiments. * p<0.05; ** p<0.01 versus the untreated cells at 20 h.

Effect of the Addition of N-Nitro-L-arginine Methyl Ester, Hydrochloride (L-NAME on HO-1 Protein Expression

We next examined whether LPS or LPS+IFN-γ-induced HO-1 protein expression in JA-4 cells would be affected by inhibiting NO production with a NOS inhibitor, L-NAME. As shown in Figs. 6A and B, JA-4 cells treated with LPS+INF-γ in the presence of L-NAME showed a significant reduction in HO-1 protein expression. In contrast, the addition of L-NAME did not alter expression of HO-1 protein significantly in the LPS-treated cells. The effect of L-NAME on NO production was similar to that on HO-1 protein expression (Fig. 6C). These results suggest that significant inhibition of iNOS seemed to be correlated with the reduction in the level of HO-1 protein in the LPS+IFN-γ-treated JA-4 cells.

Fig. 6. Inhibition of HO-1 Expression by L-NAME in LPS+IFN-γ-Treated JA-4 Cells

A: JA-4 cells were preincubated at 37°C for 17 h and then the cells were treated with LPS (100 ng/mL), INF-γ (10 units/mL) or both with or without L-NAME (1 mM) for 20 h, and the culture media were then collected and stored at −30°C until analyzed. The cell lysates were subjected to SDS-PAGE/Western blot analysis. The same membranes were probed again with an anti-β-actin antibody to ensure equal loading of cellular proteins on the gel. B: Quantification of Western blot analysis shown in “A.” C: Quantification of NO₂⁻. NO₂⁻ in the supernatants in “A” was quantified as described in the legend of Fig. 3. Error bar represents the S.E.M. for 3 independent experiments. * p<0.05; ** p<0.01 versus without L-NAME.

Induction of HO-1 Expression by NOC-12 in JA-4 and LPS1916 Cells

NO-mediated induction of HO-1 in LPS-treated macrophages was then examined by using an exogenous NO donor, NOC12, to examine whether NO was substantially involved in the regulation of HO-1 induction or not. One molecule of NOC-12 releases 2 NO molecules with a half-life of 100 min. Untreated, non-activated JA-4 and LPS1916 cells were incubated with NOC-12 (0, 50, 100 µM) for 8 h. HO-1 protein was markedly induced by NOC-12 in both JA-4 and LPS1916 cells (Figs. 7A, B). To confirm the up-regulation of HO-1 expression further, we also examined HO-1 gene expression in these cells after treatment with NOC-12. Both cells, treated with NOC-12 for longer than 2 h, showed significant and remarkable induction of HO-1 mRNA with peaks at 4 h, which expression then rapidly decreased to the basal level by 6 h (Figs. 7C, D). These results show that exogenously added NO induced HO-1 at both gene and protein expression levels.

Fig. 7. Induction of HO-1 Expression by NOC-12 in JA-4 and LPS1916 Cells

JA-4 (A) or LPS1916 (B) cells were incubated at 37°C for 17 h, and then the cells were treated with NOC12 (0, 50 or 100 µM) for 8 h. The cell lysates were subjected to Western blot analysis. The same membranes were probed again with an anti-β-actin antibody as described before. The results are presented as the means±S.E.M. for 3 independent experiments. * p<0.05; ** p<0.01 versus vehicle (0 µM NOC-12). C and D: Quantification of HO-1 mRNA levels. JA-4 (C) or LPS1916 (D) cells were incubated at 37°C for 17 h, and then the cells were treated with NOC-12 (0 [△], 50 [□] or 100 [○] µM) for the indicated times. The HO-1 gene expression was determined by using quantitative RT-PCR and normalized with the internal control GAPDH gene. The amounts are shown relative to those for the untreated cells at 0 h. Error bar represents the S.E.M. for 3 independent experiments.

Effects of the Addition of Apocynin on HO-1 Protein Expression

It is generally accepted that reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase plays a critical role in O₂⁻ generation in activated macrophages. To confirm whether O₂⁻ is also involved in induction of HO-1, we examined the effect of apocynin, which is widely used as an inhibitor of NADPH-oxidase, on HO-1 protein expression. JA-4 cells were treated without or with LPS+IFN-γ in the presence of various concentrations of apocynin for 20 h. When low concentration of apocynin (>100 µM) was used, we failed to observe an inhibitory effect of apocynin on HO-1 protein expression, O₂⁻ generation (data not shown). High concentration of apocynin (300, 400, 500 µM) significantly decreased HO-1 protein expression in LPS+IFN-γ-treated in JA-4 cells (Fig. 8A). Unexpectedly, apocynin dose-dependently inhibited not only O₂⁻ generation, but also NO production (Figs. 8B, C).

Fig. 8. Effect of Apocynin on HO-1 Protein in LPS+IFN-γ-Treated JA-4 Cells

JA-4 cells were preincubated at 37°C for 17 h. The cells were treated without (○) or with LPS+IFN-γ (▲) and apocynin (0, 300, 400 or 500 µM) for 20 h. O₂⁻ generation (A) and NO production (B) were assayed as described in Materials and Methods. C, The cell lysates were subjected to Western blot analysis. The same membranes were probed again with an anti-β-actin antibody as described before. Error bar represents the S.E.M. for 4 independent experiments. ** p<0.01 versus the untreated cells at 20 h.

Effects of the Addition of SOD and Catalase on HO-1 Protein Expression

Since, apocynin decreased both O₂⁻ generation and NO production, we next examined the effect of SOD and catalase on the induction of HO-1 protein. Because, in activated macrophages, most NADPH oxidase components assemble at the plasma membrane, and then the components produce O₂⁻ inside the phagosome or extracellularly, and most O₂⁻ is converted to H₂O₂ by nonenzymatic dismutation.^44,45) As shown in Fig. 9, JA-4 cells were incubated with LPS+IFN-γ for 8 h, and then the cells were treated further with SOD or catalase for 12 h. However, none of these treatments influenced on HO-1 protein expression in LPS+IFN-γ-treated cells, suggesting little or scarce involvement of O₂⁻ or H₂O₂ on the induction.

Fig. 9. Effects of SOD and Catalase on HO-1 Protein Expression in LPS+IFN-γ-Treated JA-4 Cells

JA-4 cells were preincubated at 37°C for 17 h. The cells were treated with LPS+IFN-γ for 8 h, and then the cells were additionally treated with SOD (50–100 µg/mL) or catalase (50–100 µg/mL) for 12 h. The cell lysates were subjected to Western blot analysis. The same membranes were probed again with an anti-β-actin antibody as described before. The results are presented as the means ±S.E.M. for 3 independent experiments.

DISCUSSION

Macrophages play essential roles in inflammation and the mobilization of the host defense against bacterial infections. LPS and IFN-γ endow macrophages with the ability to produce a wide variety of ROS, proinflammatory cytokines, and intracellular auto-protective molecules. In this study, by using the LPS-resistant mutant, LPS1916 cells, we were able to compare separately the effect of O₂⁻ and NO for HO-1 induction in LPS (or/and IFN-γ)-activated macrophages for the first time. To elucidate the regulators of HO-1 induction in activated macrophages, we examined the pattern of expression of HO-1 protein, O₂⁻-producing activity, and NO production in LPS and/or IFN-γ-treated JA-4 parental cells and LPS1916 mutant cells, whose activated macrophage phenotypes are altered.⁷⁾ Based on a comparison of the results obtained with JA-4 cells and LPS1916 cells, we found that 1) physical stimulation, such as a medium change, may have been involved in the induction of HO-1; 2) there was no close relationship between the induction of HO-1 and O₂⁻; 3) NO was the major up-regulator of HO-1 in the activated macrophages. Furthermore, the induction of HO-1 protein by NO was supported by the expression pattern of HO-1 and iNOS mRNA and protein and by the addition of L-NAME or NOC-12 to the cultures. On the other hand, we could not exclude the possibility of the involvement of the induction of HO-1 expression by other ROS, such as O₂⁻ or H₂O₂. Because LPS+IFN-γ-induced HO-1 protein was reduced more storongly than NO production by the NADPH oxidase inhibitor, apocynin (Figs. 8A, B). However, based on the results of O₂⁻ generating activity and HO-1 protein expression [i.e., LPS or IFN-γ-treated JA-4 cells and LPS+IFN-γ-treated LPS1916 cells (Fig. 2)], we consider that NO is a more important regulator of HO-1 than other ROS in LPS+IFN-γ-activated macrophages. NO is a multi-functional biomolecule involved in a variety of physiological and pathological processes, including the regulation of blood vessels; and it also has anti-arteriosclerotic, antimicrobial, and cytoprotective effects.⁴⁶⁾ It is well known that NO have a biphasic, dose-dependent effect. Low concentrations of NO have been shown to be cytoprotective against oxidative stress-induced cell death, whereas high concentrations of it are cytotoxic to many types of cells.^46,47) Our results also seemed to show the biphasic, dose-dependent effect of NO: in LPS1916 cells, although HO-1 was induced by the addition of NOC-12, the small amount of NO produced endogenously in these cells by LPS+ IFN-γ treatment at 20 h (Fig. 3B) was not able to induce HO-1 at that time (Fig. 1B). The former data indicate that HO-1 was not induced by low levels of NO produced in the cells, whereas HO-1 was strongly induced by high levels of NO either produced endogenously in the cells at 4 h (Fig. 4E) or added exogenously as NOC-12 (Fig. 6). Previous studies reported that HO-1 is induced by NO in endothelial and vascular cells^48,49) but not in macrophages. These reports concluded that HO-1 could act as a feedback inhibitor of NO when the concentrations of gaseous molecule exceed a critical threshold. Collectively, our results suggest that HO-1 may have been induced by a high, but not a low, dose of NO in the activated macrophages to protect themselves from NO-induced cytotoxicity. Furthermore, previous studies reported that HO-1 is regulated by multiple transcription factors, such as NF-E2-related factor-2 (Nrf2), activator protein-1 (AP-1), heat shock factor-1 (HSF1).^50–52) NO gas and NO donors have the potential to induce S-nitrosylation of proteins.⁵³⁾ S-Nitrosylation regulates the activity of various proteins involved in apoptosis and oxidative stress signaling. In fact, NO activates Nrf2 through S-nitrosylation of Klech-like ECH-associated protein 1 (Keap1), a key regulator of the Nrf2 signaling.⁵⁴⁾ Based on these previous data, we imply that HO-1 is induced by some transcription factor through S-nitrosylation in LPS+IFN-γ-treated macrophages. Further study is needed to clarify the molecular mechanism of NO in the induction of HO-1. To date, our data provide evidence that NO is a major up-regulator of HO-1 in LPS+IFN-γ-activated macrophages.

CONCLUSION

In activated macrophages, NO production was closely linked to the induction of HO-1, and that abolishment of NO in these cells led to a decrease in the HO-1 induction. These results suggest that NO plays an important role in HO-1 induction in activated macrophages.

Acknowledgment

This work was supported by a Grant-in-Aid for the Promotion of Science in Tokushima Knowledge Cluster Project, and a Grant-in-Aid for High Technology Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Conflict of Interest

The authors declare no conflict of interest.

REFERENCES

• 1) Aderem A, Underhill DM. Mechanisms of phagocytosis in macrophages. Annu. Rev. Immunol., 17, 593–623 (1999).
• 2) Ray A, Chattopadhyay K, Banerjee KK, Biswas T. Macrophage distinguishes Vibrio cholerae hemolysin from its protease insensitive oligomer by time dependent and selective expression of CD80–CD86. Immunol. Lett., 89, 143–147 (2003).
• 3) Akira S, Takeda K. Toll-like receptor signalling. Nat. Rev. Immunol., 4, 499–511 (2004).
• 4) Beutler B. Innate immunity: an overview. Mol. Immunol., 40, 845–859 (2004).
• 5) Akira S, Uematsu S, Takeuchi O. Pathogen recognition and innate immunity. Cell, 124, 783–801 (2006).
• 6) Bedard K, Krause KH. The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiol. Rev., 87, 245–313 (2007).
• 7) Amano F, Akamatsu Y. A lipopolysaccharide (LPS)-resistant mutant isolated from a macrophage-like cell line, J774.1, exhibits an altered activated-macrophage phenotype in response to LPS. Infect. Immun., 59, 2166–2174 (1991).
• 8) Le Page C, Sanceau J, Drapier JC, Wietzerbin J. Differential expression of inducible NO synthase in two murine macrophage cell lines. Immunology, 89, 274–280 (1996).
• 9) Nathan C. Nitric oxide as a secretory product of mammalian cells. FASEB J., 6, 3051–3064 (1992).
• 10) Nathan C, Xie QW. Regulation of biosynthesis of nitric oxide. J. Biol. Chem., 269, 13725–13728 (1994).
• 11) Assreuy J, Cunha FQ, Epperlein M, Noronha-Dutra A, O’Donnell CA, Liew FY, Moncada S. Production of nitric oxide and superoxide by activated macrophages and killing of Leishmania major. Eur. J. Immunol., 24, 672–676 (1994).
• 12) Keyse SM, Tyrrell RM. Heme oxygenase is the major 32-kDa stress protein induced in human skin fibroblasts by UVA radiation, hydrogen peroxide, and sodium arsenite. Proc. Natl. Acad. Sci. U.S.A., 86, 99–103 (1989).
• 13) Maines MD. Heme oxygenase: function, multiplicity, regulatory mechanisms, and clinical applications. FASEB J., 2, 2557–2568 (1988).
• 14) Siow RC, Sato H, Mann GE. Heme oxygenase-carbon monoxide signalling pathway in atherosclerosis: anti-atherogenic actions of bilirubin and carbon monoxide? Cardiovasc. Res., 41, 385–394 (1999).
• 15) Hayashi S, Omata Y, Sakamoto H, Higashimoto Y, Hara T, Sagara Y, Noguchi M. Characterization of rat heme oxygenase-3 gene. Implication of processed pseudogenes derived from heme oxygenase-2 gene. Gene, 336, 241–250 (2004).
• 16) Wu L, Wang R. Carbon monoxide: endogenous production, physiological functions, and pharmacological applications. Pharmacol. Rev., 57, 585–630 (2005).
• 17) Camhi SL, Lee P, Choi AM. The oxidative stress response. New Horiz., 3, 170–182 (1995).
• 18) Jaiswal AK. Antioxidant response element. Biochem. Pharmacol., 48, 439–444 (1994).
• 19) Maines MD. The heme oxygenase system: a regulator of second messenger gases. Annu. Rev. Pharmacol. Toxicol., 37, 517–554 (1997).
• 20) Ryter SW, Tyrrell RM. The heme synthesis and degradation pathways: role in oxidant sensitivity. Heme oxygenase has both pro- and anti-oxidant properties. Free Radic. Biol. Med., 28, 289–309 (2000).
• 21) Motterlini R, Foresti R, Intaglietta M, Winslow RM. NO-mediated activation of heme oxygenase: endogenous cytoprotection against oxidative stress to endothelium. Am. J. Physiol., 270, H107–H114 (1996).
• 22) Song Y, Shi Y, Ao LH, Harken AH, Meng XZ. TLR4 mediates LPS-induced HO-1 expression in mouse liver: role of TNF-alpha and IL-1beta. World J. Gastroenterol., 9, 1799–1803 (2003).
• 23) Rushworth SA, Chen XL, Mackman N, Ogborne RM, O’Connell MA. Lipopolysaccharide-induced heme oxygenase-1 expression in human monocytic cells is mediated via Nrf2 and protein kinase C. J. Immunol., 175, 4408–4415 (2005).
• 24) Camhi SL, Alam J, Otterbein L, Sylvester SL, Choi AM. Induction of heme oxygenase-1 gene expression by lipopolysaccharide is mediated by AP-1 activation. Am. J. Respir. Cell Mol. Biol., 13, 387–398 (1995).
• 25) Poss KD, Tonegawa S. Reduced stress defense in heme oxygenase 1-deficient cells. Proc. Natl. Acad. Sci. U.S.A., 94, 10925–10930 (1997).
• 26) Tracz MJ, Juncos JP, Grande JP, Croatt AJ, Ackerman AW, Rajagopalan G, Knutson KL, Badley AD, Griffin MD, Alam J, Nath KA. Renal hemodynamic, inflammatory, and apoptotic responses to lipopolysaccharide in HO-1^−/− mice. Am. J. Pathol., 170, 1820–1830 (2007).
• 27) Srisook K, Kim C, Cha YN. Cytotoxic and cytoprotective actions of O₂⁻ and NO (ONOO⁻) are determined both by cellular GSH level and HO activity in macrophages. Methods Enzymol., 396, 414–424 (2005).
• 28) Amano F, Tsukabe S, Teshima R, Waku K, Kohama K. Characterization of macrophage mutants established by their resistance to LPS and cycloheximide-induced apopotic cell death. Adv. Biosci. Biotechnol., 03, 770–781 (2012).
• 29) Inoue S, Itagaki S, Amano F. Intracellular killing of Listeria monocytogenes in the J774.1 macrophage-like cell line and the lipopolysaccharide (LPS)-resistant mutant LPS1916 cell line defective in the generation of reactive oxygen intermediates after LPS treatment. Infect. Immun., 63, 1876–1886 (1995).
• 30) Amano F, Karahashi H. A cytotoxic effect of lipopolysaccharide on a macrophage-like cell line, J774.1, in the presence of cycloheximide. J. Endotoxin Res., 3, 415–423 (1996).
• 31) Amano F, Nishijima M, Akagawa K, Akamatsu Y. Enhancement of O₂⁻ generation and tumoricidal activity of murine macrophages by a monosaccharide precursor of Escherichia coli lipid A. FEBS Lett., 192, 263–266 (1985).
• 32) Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193, 265–275 (1951).
• 33) Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680–685 (1970).
• 34) Elbirt KK, Bonkovsky HL. Heme oxygenase: recent advances in understanding its regulation and role. Proc. Assoc. Am. Physicians, 111, 438–447 (1999).
• 35) Dennery PA. Regulation and role of heme oxygenase in oxidative injury. Curr. Top. Cell. Regul., 36, 181–199 (2001).
• 36) Masuda A, Longo DL, Kobayashi Y, Appella E, Oppenheim JJ, Matsushima K. Induction of mitochondrial manganese superoxide dismutase by interleukin 1. FASEB J., 2, 3087–3091 (1988).
• 37) Visner GA, Chesrown SE, Monnier J, Ryan US, Nick HS. Regulation of manganese superoxide dismutase: IL-1 and TNF induction in pulmonary artery and microvascular endothelial cells. Biochem. Biophys. Res. Commun., 188, 453–462 (1992).
• 38) Dougall WC, Nick HS. Manganese superoxide dismutase: a hepatic acute phase protein regulated by interleukin-6 and glucocorticoids. Endocrinology, 129, 2376–2384 (1991).
• 39) Wong GH, Goeddel DV. Induction of manganous superoxide dismutase by tumor necrosis factor: possible protective mechanism. Science, 242, 941–944 (1988).
• 40) Visner GA, Dougall WC, Wilson JM, Burr IA, Nick HS. Regulation of manganese superoxide dismutase by lipopolysaccharide, interleukin-1, and tumor necrosis factor. Role in the acute inflammatory response. J. Biol. Chem., 265, 2856–2864 (1990).
• 41) Harris CA, Derbin KS, Hunte-McDonough B, Krauss MR, Chen KT, Smith DM, Epstein LB. Manganese superoxide dismutase is induced by IFN-gamma in multiple cell types. Synergistic induction by IFN-gamma and tumor necrosis factor or IL-1. J. Immunol., 147, 149–154 (1991).
• 42) Zelko IN, Mariani TJ, Folz RJ. Superoxide dismutase multigene family: a comparison of the CuZn-SOD (SOD1), Mn-SOD (SOD2), and EC-SOD (SOD3) gene structures, evolution, and expression. Free Radic. Biol. Med., 33, 337–349 (2002).
• 43) Bogdan C. Nitric oxide and the immune response. Nat. Immunol., 2, 907–916 (2001).
• 44) DeCoursey TE. Voltage-gated proton channels find their dream job managing the respiratory burst in phagocytes. Physiology, 25, 27–40 (2010).
• 45) Forman HJ, Torres M. Reactive oxygen species and cell signaling: respiratory burst in macrophage signaling. Am. J. Respir. Crit. Care Med., 166 (Suppl. 1), S4–S8 (2002).
• 46) Gotoh T, Mori M. Nitric oxide and endoplasmic reticulum stress. Arterioscler. Thromb. Vasc. Biol., 26, 1439–1446 (2006).
• 47) Chung HT, Pae HO, Choi BM, Billiar TR, Kim YM. Nitric oxide as a bioregulator of apoptosis. Biochem. Biophys. Res. Commun., 282, 1075–1079 (2001).
• 48) Naughton P, Foresti R, Bains SK, Hoque M, Green CJ, Motterlini R. Induction of heme oxygenase 1 by nitrosative stress. A role for nitroxyl anion. J. Biol. Chem., 277, 40666–40674 (2002).
• 49) Foresti R, Hoque M, Bains S, Green CJ, Motterlini R. Haem and nitric oxide: synergism in the modulation of the endothelial haem oxygenase-1 pathway. Biochem. J., 372, 381–390 (2003).
• 50) Sun J, Brand M, Zenke Y, Tashiro S, Groudine M, Igarashi K. Heme regulates the dynamic exchange of Bach1 and NF-E2-related factors in the Maf transcription factor network. Proc. Natl. Acad. Sci. U.S.A., 101, 1461–1466 (2004).
• 51) Koizumi S, Gong P, Suzuki K, Murata M. Cadmium-responsive element of the human heme oxygenase-1 gene mediates heat shock factor 1-dependent transcriptional activation. J. Biol. Chem., 282, 8715–8723 (2007).
• 52) Harada H, Sugimoto R, Watanabe A, Taketani S, Okada K, Warabi E, Siow R, Itoh K, Yamamoto M, Ishii T. Differential roles for Nrf2 and AP-1 in upregulation of HO-1 expression by arsenite in murine embryonic fibroblasts. Free Radic. Res., 42, 297–304 (2008).
• 53) Stamler JS, Lamas S, Fang FC. Nitrosylation: The prototypic redox-based signaling mechanism. Cell, 106, 675–683 (2001).
• 54) Um HC, Jang JH, Kim DH, Lee C, Surh YJ. Nitric oxide activates Nrf2 through S-nitrosylation of Keap1 in PC12 cells. J. Nitric Oxide Soc., 25, 161–168 (2011).