Agents Actions 37 (1992)
0065-4299/92/040297-08 $1.50+ 0.20/0 9 1992 Birkh/iuser Verlag, Basel
Suppressive effects of E3330, a novel quinone derivative, on tumor necrosis factor- generation from monocytes and macrophages K. Miyamoto, J. Nagakawa, I. Hishinuma, K. Hirota, M. Yasuda, T. Yamanaka, K. Katayama and I. Yamatsu Tsukuba Research Laboratories,Eisai Co. Ltd., Toukoudai 5-I-3, Tsukuba-shi, Ibaraki 300-26,Japan
Abstract
E3330{(2E)-3-[5-(2,3-dimethoxy-6-methyl-l,4-benzoquinoyl)]-2-nonyl-2-propenoic acid}, a novel synthesized hepatoprotective compound, has suppressive effects on tumor necrosis factor-~ (TNF-~) genera-. tion from monocytes/macrophages in vitro. E3330 (1 100 ~tM) reduced lipopolysaccharide (LPS, 10 mg/ml or 1 I~g/ml)-induced TNF-~ generation from rat resident and Propionibacterium acnes (P. acnes)-elicited peritoneal macrophages, rat and human monocytes, rat Kupffer cells, and splenic mononuclear cells in a concentration-dependent manner. E3330 also (1 100 I~M) suppressed TNF-~ generation stimulated with egg-albumin immune complex in rat P. acnes-elicited peritoneal macrophages. Northern blot analysis showed that LPS-induced expression of TNF-~ messenger RNA (mRNA) in human blood monocytes was suppressed by E3330. These findings indicate that E3330 has a suppressive effect on TNF-~ generation from monocytes/macrophages, regardless of origin or species, and this effect is based in part on the suppression of TNF-~ mRNA expression.
Introduction
Tumor necrosis factor-~ (TNF-e) has been recently suggested to play an important role in the pathogenesis of human liver injury, such as fulminant hepatic failure, alcoholic hepatitis, and chronic liver disease [1 3]. This idea is supported by the finding that TNF-~ production by monocytes from the patients with these conditions is significantly higher than in healthy volunteers [2, 3]. In animal models, TNF-~ is also suggested to be a key mediator of liver injury in lipopolysaccharide (LPS)-mediated hepatitis, such as galactosamine/LPS hepatitis and Propionibacterium aches ( P. acnes)-LPS hepatitis [4 6].
E3330, {(2E)-3-[5-(2,3-dimethoxy-6-methyl- 1,4benzoquinoyl)]-2-nonyl-2-propenoic acid} (Fig. 1), a newly synthesized quinone derivative has been reported as a protective agent against activated macrophage-mediated hepatitis models from our laboratory [7]. From the evidence that E3330 attenuated the elevation of circulating TNF-c~ level in the P. acnes-LPS hepatitis model, it is hypothesized that the suppressive effect of E3330 on TNF-e generation contributes to the attenuation of plasma TNF-c~ level and the hepatoprotective effects. The purpose of this study was to investigate the suppressive effects of E3330 on TNF-e generation from the several types of monocytes/macrophages
Agents Actions37 (1992)
298 O
CH30,'~-~'~'COOH o Figure l
Chemical structure of E3330 {(2E)-3-[5-(2,3-dimethoxy6-methyl-l,4-benzoquinoyl)]-2-nonyl-2-propenoicacid} (MW 378.47). in culture. Furthermore, to determine the site of suppression by E3330 on TNF-~ generation, we investigated the effect of E3330 on TNF-~ mRNA expression in human monocytes using Northern blot analysis. Materials and methods Animals
Seven-week-old Male Fisher 344 rats (weighing 180-220 g) and 5-week-old Balb/c mice (weighing 20 25 g) were obtained from Charles River Japan Inc. (Atsugi, Japan). Animals were housed at a constant temperature (23+_1~ and humidity (55% _+5%) with a fixed light-dark cycle (12-12 h) and provided with standard chow pellets and water ad libitum. Materials
E3330 was synthesized in Eisai Tsukuba Research Laboratories. P. acnes (ATCC 6918) was donated by Gifu University. Other materials were purchased commercially: Salmonella enteritidis LPS (Difco Laboratories Inc., Detroit, MI), fetal calf serum (FCS), phosphate-buffered saline (PBS) and RPMI 1640 medium (GIBCO Laboratories Inc., Gland Island, NY), type IV collagenase, pronase E, metrizamide, egg albumin and polymyxin B (Sigma, St. Louis, MO), nick-translation kits and T4-polynucleotide kinase (Takara, Kyoto, Japan), nylon membrane (Amersham Japan Co., Ltd.), Ficol-Paque (Pharmacia, Uppsala, Sweden), antiegg albumin IgG fraction (Cappel Products, Durham, NC), recombinant murine TNF-~ (Genzyme Co., Boston, MA). Isolation of various cells
Two types of peritoneal macrophages were harvested from normal rats and P. acnes-primed rats, as
reported previously [8]. The macrophages population was more than 95% as estimated by nonspecific esterase staining [9]. Human monocytes were obtained from blood of healthy donors as described previously [10]. In brief, the blood was diluted 1:1 with PBS and layered as 35 ml aliquots onto 15 ml Ficol-Paque. Following centrifugation at 400 x 9 for 30 rain, the cells at the interface were collected, diluted with PBS, and washed twice to remove remaining Ficol, and the adherent monocytes on the plastic culture plate were used. Rat monocytes were obtained from rat whole blood. Erythrocytes were sedimentated with 0.9% sodium chloride (NaC1)-6% dextran (1 g, 60 min, 25 ~C). Supernatant containing monocytes was collected and contaminating erythrocytes were lysed with hypotonic saline, and monocytes were purified by adherence to plastic culture plates. The monocytes population was more than 90% as estimated by nonspecific esterase staining [9]. Kupffer cells were isolated from rats liver by collagenase-pronase E perfusion according to the method described by Knook with slight modification [11]. In brief, the liver was perfused in situ through the portal vein with Hank's balanced salt solution (HBSS) at 37~ for 10 rain at a rate of 10ml/min. Subsequently, the liver was perfused with 0.15% pronase E in HBSS for 2 rain, and then 0.02% pronase E and 0.05% collagenase in HBSS for 15 min at flow rate of 10 ml/min at 37~ The liver was then moved and placed in the culture bottle, and was stirred in 100 ml of HBSS in the presence of 0.02% pronase and 0.05% collagenase on a magnetic stirrer at 37 ~ for 30 min. Sinusoidal cells were harvested from the digested liver by centrifugation (400 x 9, 10 min). Sinusoidal cell suspensions were freed of erythrocytes and cell debris by density centrifugation in HBSS without NaC1 containing 17.5% (W/V) metrizamide. After centrifuged at 1400 x 9 for 20 min, the cells in the top layer were washed with HBSS three times at 400 x 9 for 10 min, and they were seeded into plastic culture plate. Cells adherent to plastic plate were used as Kupffer cells. The Kupffer cell population was more than 80% as estimated by nonspecific esterase staining [9]. Mouse splenic mononuclear cells were obtained by adherence to the plastic plate. Mouse whole spleens, passed through a steel mash, was layered onto Ficol-Paque. Following centrifugation at 400 x 9 for 30 min, the cells at the interface were
Agents Actions37 (1992) diluted with PBS, washed twice, and then seeded into culture plates.
Cell culture and TNF generation The washed monocytes/macrophages were suspended in culture medium containing R P M I 1640 nutrient mixture, 10% heat-inactivated FCS, 2 m M glutamine, 100 units/ml of penicillin, and 100 tag/ml of streptomycin and seeded into 48-well culture plates (Costar| Cambridge MA) at a concentration of 1 x l06 cells/ml in a humidified 5% C O z - 9 5 % air atmosphere. The cells were incubated with 10ng/ml or 1 ~tg/ml of LPS in the presence or absence of various concentrations of E3330 at 37~ for 3-18 h. E3330 was dissolved in ethanol and added to the cells (final concentration of ethanol: 0.1%): At the end of incubation, the cellfree culture medium was collected by centrifugation (400• 10min) and stored at - 8 0 ~ prior to T N F assay. Egg-albumin immune complex was prepared according to the following method. Egg albumin and anti-egg albumin lgG fraction ( 1 : 1) were incubated for 30 min at 37~ and then washed with PBS three times. Egg-albumin immune complex thus prepared was diluted 1:20 with the medium and used for the following experiment.
299
Preparation of RNA and Northern analysis To evaluate the expression o f T N F gene, total RNA was extracted from LPS-stimulated human blood monocytes (5 • 107 cells) and investigated by Northern blot analysis. After 3 h of incubation in the presence or absence of E3330 (10 and 100~tM), total cellular RNA was isolated from cells by guanidium-isothiocyanate extraction [15] followed by cesium chloride density gradient centrifugation [16]. A 36 mer synthetic oligonucleotide, 5'-GGTGTG-GGT-GAG-GAG-CAC-ATG-GGT-GGAG G G - G C A - G C C - Y , corresponding to nucleotides 816 to 851 of human TNF-~, was synthesized. The probe was radiolabeled by [ 7 - 3 2 p ] ATP and T4-polynucleotide kinase. The resultant specific activity was 1 to 2 x l 0 8 c p m / m g . The concentration of the RNA was determined by spectrophotometry at 260 and 280nm. The RNA was subjected to electrophoresis in a 1% agarose formaldehyde gel, and was then transferred onto nylon membranes and hybridized as described [17]. The membranes were washed four times for 10 rain at room temperature with 0.1% SDS-6 x standard sodium citrate (SSC; 1 x SSC =0.15 M NaC1 and 0.015 M sodium citrate), followed by further twice washes with 0.1% SDS-6 x SSC for 30 min at 55~ The membranes were exposed to Kodak XAR-5 X-ray film for 1 3 days at - 7 0 ~ using an intensifying screen. Membranes which equivalent amounts of RNA were blotted were analyzed with the probe for fl-actin as described above [18].
Assay of TNF TNF-c~ titer in culture medium was determined by the cytotoxicity of highly TNF-sensitive L-P3 cells, a subline of L-929 cells [12], in the presence of 1 ~tg/ml actinomycin D, as described previously [13]. In brief, the culture samples and standard were prediluted in culture medium. Then 50 jal of the prediluted samples or standard recombinant murine TNF-c~ was added to 96-wells multiplates, and serially diluted (2-fold dilution ratio) with the culture medium. Subsequently, 50~tl of 7 x 105 cells/ml cell suspension of L-P3, containing 2 ~tg/ml of actinomycin D, was added, and the plate was incubated for 12-15 h at 37~ in a humidified 5% CO2 95% air atmosphere. The dilution ratio associated with 50% cytotoxicity was evaluated by the methylene blue staining method [14]. One mg of recombinant murine TNF-~ corresponded to 3 x 10 9 laboratory units.
Statistical analysis All data were expressed as the mean values • standard error and were statistically analyzed by Williams Wilcoxon test. Differences with p
0065-4299/92/040297-08 $1.50+ 0.20/0 9 1992 Birkh/iuser Verlag, Basel
Suppressive effects of E3330, a novel quinone derivative, on tumor necrosis factor- generation from monocytes and macrophages K. Miyamoto, J. Nagakawa, I. Hishinuma, K. Hirota, M. Yasuda, T. Yamanaka, K. Katayama and I. Yamatsu Tsukuba Research Laboratories,Eisai Co. Ltd., Toukoudai 5-I-3, Tsukuba-shi, Ibaraki 300-26,Japan
Abstract
E3330{(2E)-3-[5-(2,3-dimethoxy-6-methyl-l,4-benzoquinoyl)]-2-nonyl-2-propenoic acid}, a novel synthesized hepatoprotective compound, has suppressive effects on tumor necrosis factor-~ (TNF-~) genera-. tion from monocytes/macrophages in vitro. E3330 (1 100 ~tM) reduced lipopolysaccharide (LPS, 10 mg/ml or 1 I~g/ml)-induced TNF-~ generation from rat resident and Propionibacterium acnes (P. acnes)-elicited peritoneal macrophages, rat and human monocytes, rat Kupffer cells, and splenic mononuclear cells in a concentration-dependent manner. E3330 also (1 100 I~M) suppressed TNF-~ generation stimulated with egg-albumin immune complex in rat P. acnes-elicited peritoneal macrophages. Northern blot analysis showed that LPS-induced expression of TNF-~ messenger RNA (mRNA) in human blood monocytes was suppressed by E3330. These findings indicate that E3330 has a suppressive effect on TNF-~ generation from monocytes/macrophages, regardless of origin or species, and this effect is based in part on the suppression of TNF-~ mRNA expression.
Introduction
Tumor necrosis factor-~ (TNF-e) has been recently suggested to play an important role in the pathogenesis of human liver injury, such as fulminant hepatic failure, alcoholic hepatitis, and chronic liver disease [1 3]. This idea is supported by the finding that TNF-~ production by monocytes from the patients with these conditions is significantly higher than in healthy volunteers [2, 3]. In animal models, TNF-~ is also suggested to be a key mediator of liver injury in lipopolysaccharide (LPS)-mediated hepatitis, such as galactosamine/LPS hepatitis and Propionibacterium aches ( P. acnes)-LPS hepatitis [4 6].
E3330, {(2E)-3-[5-(2,3-dimethoxy-6-methyl- 1,4benzoquinoyl)]-2-nonyl-2-propenoic acid} (Fig. 1), a newly synthesized quinone derivative has been reported as a protective agent against activated macrophage-mediated hepatitis models from our laboratory [7]. From the evidence that E3330 attenuated the elevation of circulating TNF-c~ level in the P. acnes-LPS hepatitis model, it is hypothesized that the suppressive effect of E3330 on TNF-e generation contributes to the attenuation of plasma TNF-c~ level and the hepatoprotective effects. The purpose of this study was to investigate the suppressive effects of E3330 on TNF-e generation from the several types of monocytes/macrophages
Agents Actions37 (1992)
298 O
CH30,'~-~'~'COOH o Figure l
Chemical structure of E3330 {(2E)-3-[5-(2,3-dimethoxy6-methyl-l,4-benzoquinoyl)]-2-nonyl-2-propenoicacid} (MW 378.47). in culture. Furthermore, to determine the site of suppression by E3330 on TNF-~ generation, we investigated the effect of E3330 on TNF-~ mRNA expression in human monocytes using Northern blot analysis. Materials and methods Animals
Seven-week-old Male Fisher 344 rats (weighing 180-220 g) and 5-week-old Balb/c mice (weighing 20 25 g) were obtained from Charles River Japan Inc. (Atsugi, Japan). Animals were housed at a constant temperature (23+_1~ and humidity (55% _+5%) with a fixed light-dark cycle (12-12 h) and provided with standard chow pellets and water ad libitum. Materials
E3330 was synthesized in Eisai Tsukuba Research Laboratories. P. acnes (ATCC 6918) was donated by Gifu University. Other materials were purchased commercially: Salmonella enteritidis LPS (Difco Laboratories Inc., Detroit, MI), fetal calf serum (FCS), phosphate-buffered saline (PBS) and RPMI 1640 medium (GIBCO Laboratories Inc., Gland Island, NY), type IV collagenase, pronase E, metrizamide, egg albumin and polymyxin B (Sigma, St. Louis, MO), nick-translation kits and T4-polynucleotide kinase (Takara, Kyoto, Japan), nylon membrane (Amersham Japan Co., Ltd.), Ficol-Paque (Pharmacia, Uppsala, Sweden), antiegg albumin IgG fraction (Cappel Products, Durham, NC), recombinant murine TNF-~ (Genzyme Co., Boston, MA). Isolation of various cells
Two types of peritoneal macrophages were harvested from normal rats and P. acnes-primed rats, as
reported previously [8]. The macrophages population was more than 95% as estimated by nonspecific esterase staining [9]. Human monocytes were obtained from blood of healthy donors as described previously [10]. In brief, the blood was diluted 1:1 with PBS and layered as 35 ml aliquots onto 15 ml Ficol-Paque. Following centrifugation at 400 x 9 for 30 rain, the cells at the interface were collected, diluted with PBS, and washed twice to remove remaining Ficol, and the adherent monocytes on the plastic culture plate were used. Rat monocytes were obtained from rat whole blood. Erythrocytes were sedimentated with 0.9% sodium chloride (NaC1)-6% dextran (1 g, 60 min, 25 ~C). Supernatant containing monocytes was collected and contaminating erythrocytes were lysed with hypotonic saline, and monocytes were purified by adherence to plastic culture plates. The monocytes population was more than 90% as estimated by nonspecific esterase staining [9]. Kupffer cells were isolated from rats liver by collagenase-pronase E perfusion according to the method described by Knook with slight modification [11]. In brief, the liver was perfused in situ through the portal vein with Hank's balanced salt solution (HBSS) at 37~ for 10 rain at a rate of 10ml/min. Subsequently, the liver was perfused with 0.15% pronase E in HBSS for 2 rain, and then 0.02% pronase E and 0.05% collagenase in HBSS for 15 min at flow rate of 10 ml/min at 37~ The liver was then moved and placed in the culture bottle, and was stirred in 100 ml of HBSS in the presence of 0.02% pronase and 0.05% collagenase on a magnetic stirrer at 37 ~ for 30 min. Sinusoidal cells were harvested from the digested liver by centrifugation (400 x 9, 10 min). Sinusoidal cell suspensions were freed of erythrocytes and cell debris by density centrifugation in HBSS without NaC1 containing 17.5% (W/V) metrizamide. After centrifuged at 1400 x 9 for 20 min, the cells in the top layer were washed with HBSS three times at 400 x 9 for 10 min, and they were seeded into plastic culture plate. Cells adherent to plastic plate were used as Kupffer cells. The Kupffer cell population was more than 80% as estimated by nonspecific esterase staining [9]. Mouse splenic mononuclear cells were obtained by adherence to the plastic plate. Mouse whole spleens, passed through a steel mash, was layered onto Ficol-Paque. Following centrifugation at 400 x 9 for 30 min, the cells at the interface were
Agents Actions37 (1992) diluted with PBS, washed twice, and then seeded into culture plates.
Cell culture and TNF generation The washed monocytes/macrophages were suspended in culture medium containing R P M I 1640 nutrient mixture, 10% heat-inactivated FCS, 2 m M glutamine, 100 units/ml of penicillin, and 100 tag/ml of streptomycin and seeded into 48-well culture plates (Costar| Cambridge MA) at a concentration of 1 x l06 cells/ml in a humidified 5% C O z - 9 5 % air atmosphere. The cells were incubated with 10ng/ml or 1 ~tg/ml of LPS in the presence or absence of various concentrations of E3330 at 37~ for 3-18 h. E3330 was dissolved in ethanol and added to the cells (final concentration of ethanol: 0.1%): At the end of incubation, the cellfree culture medium was collected by centrifugation (400• 10min) and stored at - 8 0 ~ prior to T N F assay. Egg-albumin immune complex was prepared according to the following method. Egg albumin and anti-egg albumin lgG fraction ( 1 : 1) were incubated for 30 min at 37~ and then washed with PBS three times. Egg-albumin immune complex thus prepared was diluted 1:20 with the medium and used for the following experiment.
299
Preparation of RNA and Northern analysis To evaluate the expression o f T N F gene, total RNA was extracted from LPS-stimulated human blood monocytes (5 • 107 cells) and investigated by Northern blot analysis. After 3 h of incubation in the presence or absence of E3330 (10 and 100~tM), total cellular RNA was isolated from cells by guanidium-isothiocyanate extraction [15] followed by cesium chloride density gradient centrifugation [16]. A 36 mer synthetic oligonucleotide, 5'-GGTGTG-GGT-GAG-GAG-CAC-ATG-GGT-GGAG G G - G C A - G C C - Y , corresponding to nucleotides 816 to 851 of human TNF-~, was synthesized. The probe was radiolabeled by [ 7 - 3 2 p ] ATP and T4-polynucleotide kinase. The resultant specific activity was 1 to 2 x l 0 8 c p m / m g . The concentration of the RNA was determined by spectrophotometry at 260 and 280nm. The RNA was subjected to electrophoresis in a 1% agarose formaldehyde gel, and was then transferred onto nylon membranes and hybridized as described [17]. The membranes were washed four times for 10 rain at room temperature with 0.1% SDS-6 x standard sodium citrate (SSC; 1 x SSC =0.15 M NaC1 and 0.015 M sodium citrate), followed by further twice washes with 0.1% SDS-6 x SSC for 30 min at 55~ The membranes were exposed to Kodak XAR-5 X-ray film for 1 3 days at - 7 0 ~ using an intensifying screen. Membranes which equivalent amounts of RNA were blotted were analyzed with the probe for fl-actin as described above [18].
Assay of TNF TNF-c~ titer in culture medium was determined by the cytotoxicity of highly TNF-sensitive L-P3 cells, a subline of L-929 cells [12], in the presence of 1 ~tg/ml actinomycin D, as described previously [13]. In brief, the culture samples and standard were prediluted in culture medium. Then 50 jal of the prediluted samples or standard recombinant murine TNF-c~ was added to 96-wells multiplates, and serially diluted (2-fold dilution ratio) with the culture medium. Subsequently, 50~tl of 7 x 105 cells/ml cell suspension of L-P3, containing 2 ~tg/ml of actinomycin D, was added, and the plate was incubated for 12-15 h at 37~ in a humidified 5% CO2 95% air atmosphere. The dilution ratio associated with 50% cytotoxicity was evaluated by the methylene blue staining method [14]. One mg of recombinant murine TNF-~ corresponded to 3 x 10 9 laboratory units.
Statistical analysis All data were expressed as the mean values • standard error and were statistically analyzed by Williams Wilcoxon test. Differences with p
