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. 2011 Nov;166(2):299–306. doi: 10.1111/j.1365-2249.2011.04475.x

Dehydroxymethylepoxyquinomicin (DHMEQ) can suppress tumour necrosis factor-α production in lipopolysaccharide-injected mice, resulting in rescuing mice from death in vivo

T Shimo *, Y Adachi †,‡, K Umezawa §, M Okigaki , J Takaya *, S Taniuchi *, S Ikehara ‡, K Kaneko *
PMCID: PMC3219905  PMID: 21985376

Abstract

Dehydroxymethylepoxyquinomicin (DHMEQ), a new nuclear factor (NF)-κB inhibitor, has several beneficial effects, including the suppression of tumour growth and anti-inflammatory effects. DHMEQ can also suppress the production of tumour necrosis factor (TNF)-α induced by lipopolysaccharide (LPS) in vitro. In the present study, we examine the effects of DHMEQ on TNF-α production in vivo and on the survival of mice injected with LPS. When DHMEQ was injected into mice 2 h before LPS injection, the survival of the LPS-injected mice was prolonged. When DHMEQ was injected twice (2 h before LPS injection and the day after LPS injection), all the mice were rescued. The injection of DHMEQ 1 h after LPS injection and the day after LPS injection also resulted in the rescue of all mice. The serum levels of TNF-α in the mice that received both LPS and DHMEQ were suppressed compared to the mice that received only LPS. These results suggest that DHMEQ can be utilized for the prevention and treatment of endotoxin shock.

Keywords: dehydroxymethylepoxyquinomicin (DHMEQ), in vivo, LPS, mice, TNF-α

Introduction

It is currently believed that endotoxin shock is caused by inflammation-induced hypercytokinaemia [1,2]. In endotoxin shock induced by Gram-negative rods, lipopolysaccharide (LPS) stimulates inflammatory cells to release cytokines such as tumour necrosis factor (TNF)-α[3]. TNF-α is an apoptosis-inducing factor [1,4,5], as the TNF-α receptor contains a death domain, such as Fas [6–8]. We found previously that anti-TNF-α antibody can rescue LPS-injected mice from death [9]. Thus, it appears that TNF-α is one of the most critical cytokines in endotoxin shock. We have also found that the vulnerability of (NZW × BXSB) F1 mice (W/BF1 mice) to LPS is greater than that of normal mice and that this increased vulnerability is the result of enhanced TNF-α production [9]. Not only monocyte/macrophage lineage cells but also dendritic cells contribute to TNF-α production in W/BF1 mice.

Dehydroxymethylepoxyquinomicin (DHMEQ) is a new nuclear factor (NF)-κB inhibitor [10]. The DHMEQ-mediated inhibition of NF-κB is potent and results in several interesting effects: DHMEQ can reduce atherosclerosis in apolipoprotein E (ApoE)-knock-out mice [11] and suppress the growth of prostate cancer [12,13], bladder cancer [14], thyroid cancer [15], multiple myeloma [16], breast cancer [17], adult T cell leukaemia [18–20], pancreatic cancer [21] and neck squamous cell carcinoma [22]; DHMEQ can also improve arthritis in rodents [23] and suppress anti-Thy1·1-induced glomerulonephritis in rats [24].

DHMEQ can suppress TNF-α production in microglia cells and macrophages in vitro[10,25], and DHMEQ can be administered safely in vivo to rodents [11,12,15–20]. However, the in vivo effects of DHMEQ on TNF-α production have not been elucidated. In the present study, we show the suppressive effects of DHMEQ on LPS-induced TNF-α production in vivo, resulting in the rescue of LPS-injected mice from death.

Materials and methods

Mice

Male C57BL/6J mice (B6 mice) and male W/BF1 mice were purchased from SLC (Shizuoka, Japan). Male W/BF1 mice more than 12 weeks old that exhibited more than (++) proteinuria (Albustix®, Bayer Medical Ltd, Tokyo, Japan) and 12-week-old male B6 mice were used for this experiment.

Chemicals

LPS (Sigma Chemical Co., St Louis, MO, USA) was injected into the peritoneal cavity of mice. DHMEQ, which was synthesized by Umezawa et al. [26], was dissolved in dimethylsulphoxide (DMSO) (Nacalai Tesque, Kyoto, Japan) to prepare a 10 µg/ml stock solution, and stored at −20°C until use. For in vitro culture experiments, the DHMEQ in DMSO was diluted with culture medium. For culture without DHMEQ, an equivalent volume of DMSO was added to the culture medium, except for the samples shown in Fig. 1c. For in vivo experiments, just before injection of DHMEQ, the DHMEQ in DMSO was diluted with RPMI-1640 (Nikken Bio Medical Laboratory, Kuzeyama-cho, Kyoto, Japan) to a final concentration of less than 1% DMSO. The mice without DHMEQ were injected with the same volume of DMSO in RPMI-1640.

Fig. 1.

Fig. 1

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Dehydroxymethylepoxyquinomicin (DHMEQ) can suppress lipopolysaccharide (LPS)-induced tumour necrosis factor (TNF)-α production in vitro. (a) A single-cell suspension of spleen cells was obtained from B6 mice and the cells were suspended in RPMI-1640 with 10% fetal calf serum (FCS). DHMEQ and/or LPS were added and the cells were cultured for 18 h, before the supernatant was obtained for measurement of the TNF-α concentration (n = 4, *P < 0·05). (b) Spleen cells were cultured for 0·5 h or 2 h with or without DHMEQ and/or LPS, and then reverse transcription–polymerase chain reaction (RT–PCR) was carried out to examine the TNF-α mRNA expression (n = 4, *:P < 0·05). (c) Whole spleen cells or CD11b+-enriched spleen cells were cultured with or without DHMEQ and/or LPS. In this experiment, we prepared two kinds of controls: one without both dimethylsulphoxide (DMSO) and DHMEQ, and another using DMSO alone instead of DHMEQ. After 2 h culture, RNA was obtained and RT–PCR was carried out to examine the mRNA expression of TNF-α, interleukin (IL)-6 and IL-1β (n = 4, *P < 0·05). (d) After 4 h culture, the spleen cells were stained with anti-CD11b antibody, followed by permeabilization and staining with anti-TNF-α antibody (n = 4, *P < 0·05). Representative data from two independent experiments are shown in Fig. 1a–d.

Cell culture

Spleen cells or CD11b+-enriched spleen cells (2 × 106 cells/ml) from B6 mice or W/BF1 mice were cultured with or without LPS (1 µg/ml) and/or DHMEQ (10 ng/ml) in RPMI-1640 with 10% fetal calf serum (FCS) and antibiotics. CD11b+-enriched spleen cells were prepared using anti-CD11b antibody-coated MACS beads (Miltenyi Biotec, Bergisch Gladbach, Germany).

Histological analyses

B6 mice were killed 24 h after LPS injection (20 mg/kg), and their organs were fixed with buffered formalin. Haematoxylin and eosin staining and a terminal deoxynucleotidyl transferase-mediated 2′-deoxyuridine, 5′-triphosphate (dUTP) nick end labelling (TUNEL) assay were performed on the organs. The TUNEL assay was performed with an in-situ apoptosis detection kit (Takara, Otsu, Japan). To examine the localization of NF-κB in the hepatocytes, we first incubated the liver samples with anti-NF-κB antibody (Cell Signaling Technology, Danvers, MA, USA). Next, EnVision Systems (Dako, Glostrup, Denmark) was used to visualize the localization.

Reverse transcription–polymerase chain reaction (RT–PCR) to examine mRNA expression of TNF-α, interleukin (IL)-1β and IL-6 in spleen cells

The cDNA from cultured spleen cells was prepared as described previously [27]. After adjusting the quantity of cDNA, a polymerase chain reaction was performed with glyceraldehyde-3-phosphate dehydrogenase (G3PDH)-specific primers (Toyobo, Tokyo, Japan) or mouse TNF-α-specific primers, mouse IL-6-specific primers (Maxim Biotech, Inc., San Francisco, CA, USA) and mouse IL-1β-specific primers [28].

Measurement of TNF-α concentration in serum and supernatant

Serum from LPS-treated mice and supernatant from LPS-treated cultures were collected at various time-points after the treatments. The serum and supernatant were stored at −20°C until used. The TNF-α concentration was measured using an enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Inc., Minneapolis, MN, USA).

Intracytoplasmic staining for TNF-α in spleen cells

Spleen cells from B6 mice and W/BF1 mice were cultured in 10% FCS containing RPMI-1640 with LPS (1 mg/ml) with or without Brefeldin A (10 mg/ml; Sigma). After 2-h incubation, the cells were harvested and stained. First, surface staining was performed with fluorescein isothiocyanate (FITC)-labelled anti-CD11c, peridinin chlorophyll (PerCP) Cy5·5-labelled anti-CD45 and allophycocyanin (APC)-labelled anti-CD11b antibodies (BD Bioscience Pharmingen, San Jose, CA, USA). Next, cells were permeabilized with Intra-Prep® (Immunotech, Beckman Coulter Co., Marseille, France), and intracytoplasmic staining was performed with phycoerythrin (PE)-labelled anti-TNF-α antibody. The cells were analysed with BD FACScalibur™ (BD Bioscience Pharmingen).

Statistical analysis

Differences between mean values were assessed by a two-tailed t-test. P-values less than 0·05 were considered statistically significant. The rate of survival was assessed using the Kaplan–Meier test.

Results

DHMEQ inhibits TNF-α production in vitro

We and other researchers have found that the death of LPS-injected mice is caused by the overproduction of TNF-α[9,29,30] and that DHMEQ can suppress the LPS-induced TNF-α production from macrophage and microglia in vitro[25,31]. First, we confirmed the suppressive effects of DHMEQ on TNF-α production in whole spleen cells in vitro. As shown in Fig. 1a, DHMEQ inhibited the LPS-induced TNF-α production when DHMEQ was added to spleen cell cultures 2 h before the addition of LPS. Next, we examined the expression of TNF-α mRNA, and found that DHMEQ also suppressed its expression in B6 mice (Fig. 1b). It has also been reported that LPS can induce not only TNF-α but also IL-6 and IL-1 [32]. We examined whether DHMEQ can suppress mRNA expression of IL-6 and IL-1β. As shown in Fig. 1c, DHMEQ was found to suppress mRNA expression of not only TNF-α but also of IL-6 and IL-1β. DMSO was used as a vehicle for DHMEQ; therefore, a control with DMSO only at the same volume as DHMEQ was used in the culture wells. However, there were no observable effects of DMSO on the mRNA expression of the cytokines. The data in Fig. 1d show that DHMEQ can completely suppress the LPS-induced TNF-α production in the monocyte/macrophage lineage cells of B6 mice. These results suggest that DHMEQ can suppress the expression of TNF-α mRNA, resulting in the suppression of TNF-α production.

DHMEQ reduces apoptotic cells in spleens of LPS-injected mice and inhibits shift of NF-κB into nucleus

It has been reported that LPS induces apoptosis in the lymphoid organs [1]. Therefore, we examined apoptotic cells in the spleen using the TUNEL assay. As shown in Fig. 2a,b, TUNEL-positive cells were abundant in the spleens of LPS-treated mice. However, the number of TUNEL-positive cells in the spleens of LPS-treated mice was reduced significantly when the mice were pretreated with DHMEQ.

Fig. 2.

Fig. 2

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Dehydroxymethylepoxyquinomicin (DHMEQ) can suppress lipopolysaccharide (LPS)-induced apoptosis and movement of nuclear factor (NF)-κB into the nucleus in vivo. (a) B6 mice were treated with DHMEQ and/or LPS as described in Materials and methods. After 24 h, the mice were killed and apoptotic cells in the spleen were examined using the terminal deoxynucleotidyl transferase-mediated 2′-deoxyuridine, 5′-triphosphate (dUTP) nick end labelling (TUNEL) assay. Representative photographs of TUNEL staining are shown: left, spleen cells from the mice treated with LPS without DHMEQ; right, spleen cells from the mice treated with LPS and DHMEQ (apoptotic cells are coloured brown). (b) The mean and standard deviation (s.d.) of the apoptotic cell numbers per high-power field of the spleens are shown. (n = 4, *:P < 0·05) (c) Localization of NF-κB was examined by staining liver with anti-NF-κB p65 antibody. The left photograph shows the liver from mice treated with LPS without DHMEQ, while the right photograph shows the liver from mice treated with LPS and DHMEQ (NF-κB are coloured brown). Arrows show intranuclear NF-κB. Representative data from two independent experiments are shown.

We confirmed the inhibition of the NF-κB shift from the cytoplasm into the nucleus. As shown in Fig. 2c, when mice were treated with LPS, NF-κB moved into the nucleus, resulting in browning of the hepatocyte nuclei. However, when mice were treated with DHMEQ and LPS, nuclear shift of NF-κB from the cytoplasm was inhibited.

DHMEQ rescues LPS-injected mice from death

We examined the effects of DHMEQ on B6 mice in vivo. First, we examined single injections of various amounts of DHMEQ into the peritoneal cavity of B6 mice. Two hours after the injection of DHMEQ, a high dose of LPS (20 mg/kg) was injected into the peritoneal cavities of B6 mice. As shown in Fig. 3a, LPS-treated mice without DHMEQ treatment died rapidly, while LPS-treated mice with DHMEQ pretreatment showed a better survival rate. The survival rate was more effective by 20 mg/kg of DHMEQ compared with lower doses; however, more than 50% of DHMEQ-pretreated mice from each dose group eventually died.

Fig. 3.

Fig. 3

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Dehydroxymethylepoxyquinomicin (DHMEQ) can reduce mortality rate in lipopolysaccharide (LPS)-treated mice. (a) Several dosages of DHMEQ (0, 8, 10 or 20 mg/kg) were injected into mice 2 h before LPS injection. The mice were observed until 100 h after LPS-injection [numbers of mice, shown as (experimental mice/dead mice/rescued mice), for each treatment group are as follows: (8/4/4), DHMEQ 8 mg/kg DHMEQ; (8/6/2) 10 mg/kg DHMEQ; (8/5/3), 20 mg/kg DHMEQ; and (8/8/0), without DHMEQ; *P < 0·05]. (b) DHMEQ was injected for 2 consecutive days (day 0 and day 1), and various timings for the initial DHMEQ injections were tested with LPS-injected mice. Initial DHMEQ injections were performed at 2 h before LPS injection, 1 h after LPS injection, 2 h after LPS injection or 3 h after LPS injection. On the next day (day 1), the mice were injected again with DHMEQ at 24 h after LPS injection. The mice were observed until 100 h after LPS injection [numbers of mice, shown as (experimental mice/dead mice/rescued mice), for each initial injection of DHMEQ timing group are as follows: (6/0/6), 2 h before LPS injection; (6/0/6), 1 h after LPS injection; (6/4/2), 2 h after LPS injection; and (6/5/1), 3 h after LPS-injection; *P < 0·05].

Next, we injected DHMEQ twice (10 mg/kg per injection); mice received DHMEQ on the same day as LPS injection and 24 h after the first injection of DHMEQ. As shown in Fig. 3b, all mice injected with DHMEQ 2 h before or 1 h after LPS injection survived. However, DHMEQ injected at 2 or 3 h after LPS injection had no effect on the survival rate. These results suggest that DHMEQ can suppress the effect of LPS if it is injected within 1 h after LPS and that two injections of DHMEQ (the same day as LPS injection and the day after LPS injection) are necessary to suppress the effects of LPS.

DHMEQ suppresses LPS-induced TNF-α production in vivo

Next, we examined the serum concentration of TNF-α in the LPS and/or DHMEQ-injected mice. Injection of only DHMEQ had no effect on the serum concentration of TNF-α. Conversely, the injection of LPS induced an increase in the serum concentration of TNF-α, as shown in Fig. 4. However, the injection of DHMEQ at 2 h before or 1 h after LPS injection reduced the concentration of serum TNF-α, while DHMEQ injection at 2 or 3 h after LPS injection did not reduce the serum concentration of TNF-α. These results suggest that DHMEQ injection before or 1 h after LPS injection can suppress the production of TNF-α, resulting in the rescue of mice from LPS-induced death.

Fig. 4.

Fig. 4

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Dehydroxymethylepoxyquinomicin (DHMEQ) can suppress serum tumour necrosis factor (TNF)-α induced by lipopolysaccharide (LPS) in vivo. DHMEQ was injected into the mice 2 h before LPS injection, 1 h after LPS injection or 3 h after LPS injection. Serum of more than four mice in each group was obtained just before LPS injection, 2 h after LPS injection and 4 h after LPS injection (n = 4 in each group; *P < 0·05).

DHMEQ is effective even in W/BF1 mice

We examined the effects of DHMEQ in W/BF1 mice, which are highly vulnerable to LPS. When DHMEQ was injected 2 h before the LPS injection and 24 h after the first DHMEQ injection, all W/BF1 mice survived (Fig. 5a). Without DHMEQ, more than 70% of LPS-injected W/BF1 mice died.

Fig. 5.

Fig. 5

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Dehydroxymethylepoxyquinomicin (DHMEQ) can suppress tumour necrosis factor (TNF)-α production even in autoimmune-prone W/BF1 mice. (a) DHMEQ was injected into W/BF1 mice 2 h before lipopolysaccharide (LPS) injection. Survival rate up to 100 h post-LPS injection is shown [numbers of W/BF1 mice, shown as (experimental mice/dead mice/rescued mice) for each treatment group are as follows: (6/0/6), DHMEQ; and (7/5/2) without DHMEQ = 7; P < 0·05]. (b) Spleen cells of W/BF1 mice were cultured with or without DHMEQ and/or LPS for 24 h. Culture supernatant was obtained and the concentration of TNF-α was examined (n = 4; *P < 0·05). (c) DHMEQ was injected into W/BF1 mice 2 h before LPS injection. Serum from the mice was obtained 2 h after LPS injection (n = 4; *P < 0·05).

Next, we examined the effects of DHMEQ on LPS-induced TNF-α production in WBF1 mice in vitro. As shown in Fig. 5b, DHMEQ suppressed the production of TNF-α even in W/BF1 mice.

We also examined the effects of DHMEQ on TNF-α production of W/BF1 mice in vivo. As shown in Fig. 5c, two injections of DHMEQ (2 h before LPS injection and 24 h after the first injection of DHMEQ) suppressed the production of TNF-αin vivo. These results suggest that DHMEQ can suppress the production of TNF-α even in W/BF1 mice, which are highly vulnerable to LPS, resulting in the rescue of the mice from death.

Discussion

We and other researchers have found previously that in vivo injection of LPS induces TNF-α production, especially from monocyte/macrophage lineage cells, and that anti-TNF-α antibody can rescue LPS-injected mice from death [9,29,30]. Therefore, TNF-α suppression is thought to be an important strategy for the treatment of endotoxin shock caused by LPS. We chose DHMEQ for the inhibition of NF-κB because of its specificity and low toxicity in animals. It binds specifically and covalently to NF-κB components to directly inhibit DNA binding [33], and is therefore considered to be more specific than most other NFκB inhibitors. Also, it has been used in more than 20 animal experiments to date and there have been no reports of observable toxicity [34]. Because NF-κB is involved both downstream and upstream of TNF-α signalling, DHMEQ should be able to inhibit both.

DHMEQ completely inhibited the LPS-induced TNF-α production by spleen cells, both at the protein level and the mRNA level (Fig. 1), as described previously by Takatsuna et al. [10,25,31]. Moreover, we found that DHMEQ can suppress LPS-induced apoptosis in vivo (Fig. 2). These results suggest that DHMEQ has suppressive effects on LPS-induced apoptosis both in vivo and in vitro. Therefore, DHMEQ could be a new strategy for the treatment and prevention of endotoxin shock caused by LPS. Based on this concept, we administered DHMEQ to LPS-treated mice (Fig. 3). However, the suppressive effect of DHMEQ on LPS-induced mortality was limited when DHMEQ was provided as a single injection. Therefore, we administered DHMEQ as two separate injections (10 mg/kg per injection) on 2 consecutive days. When the initial DHMEQ injection was provided 2 h before LPS injection or 1 h after LPS injection, all the mice survived. However, when the initial DHMEQ injection was provided 2 or 3 h after LPS injection, the suppressive effect of DHMEQ on mortality was extremely limited. These results suggest that DHMEQ is metabolized rapidly in vivo. Therefore, DHMEQ should be injected frequently during endotoxin shock. Our results also suggest that DHMEQ injection during pre-shock status or during the early stage of endotoxin shock is necessary to rescue individuals and that delayed injection of DHMEQ has no effect. The timing of DHMEQ injection is therefore crucial for its successful use, as the survival rates were correlated with serum TNF-α concentration (Figs 3 and 4). Administration of DHMEQ 3 h after LPS injection resulted in high serum TNF-α concentration and high mortality, while administration of DHMEQ 2 h before or 1 h after LPS injection resulted in low serum TNF-α concentration and low mortality. Our previous data showed that TNF-α mRNA peaks at 2 h after LPS stimulation [9]. Therefore, it is conceivable that pre-administration of DHMEQ or administration of DHMEQ at 1 h after LPS injection can suppress TNF-α mRNA synthesis induced by LPS, resulting in the suppression of TNF-α production.

It is reported that DHMEQ can induce apoptosis in multiple myeloma cells [35], but that it can suppress LPS-induced apoptosis in the spleen cells of LPS-injected mice and can rescue the mice from death. In tumour cells, NF-κB is activated constitutively and the inhibition of NF-κB activation by DHMEQ can induce cleavage of caspase 3, resulting in the induction of apoptosis. However, NF-κB usually exists in its inactive form within the cytoplasm of normal cells. When the normal monocyte/macrophage lineage cells are treated with LPS, NF-κB is activated and moves into the nucleus where it induces the production of TNF-α mRNA and other cytokines. These cytokines induce apoptosis of various normal cells, which can lead to the death of the individual.

The results of the present study illustrate the beneficial effects of DHMEQ on LPS-treated mice. Our results suggest that multiple injections of DHMEQ can be utilized as a new strategy for the treatment of early-stage endotoxin shock and the prevention of oncoming endotoxin shock.

Acknowledgments

We thank Mr F. Kawakami, Ms H. Ogaki, Mr K. Nagaoka, Ms M. Nishimura and Ms S. Eriguchi at Public Toyooka Hospital for their expert technical assistance, and also Ms T. Katsuta in the Department of Pediatrics, Kasai Medical University, for aid in the preparation of this manuscript. This work was supported by Grant-in-Aid for Scientific Research (C) 22591125 and 21590447. This work was also supported by the Research Grant (F) from Kansai Medical University and the Mami Mizutani Foundation.

Disclosure

Authors have no disclosures to report.

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