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secretory products from plerocercoids of Spirometra erinaceieuropaei suppress gene expressions and production of tumor necrosis factor-α in murine macrophages stimulated with lipopolysaccharide or lipoteichoic acid
International Journal for Parasitology 31 (2000) 39±47
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Excretory/secretory products from plerocercoids of Spirometra erinaceieuropaei suppress gene expressions and production of tumor necrosis factor-a in murine macrophages stimulated with lipopolysaccharide or lipoteichoic acid K. Miura*, S. Fukumoto, P. Dirgahayu, K. Hirai Department of Medical Zoology, Faculty of Medicine, Tottori University, 86 Nishi-Cho, Yonago 683-8503, Japan Received 5 September 2000; received in revised form 10 November 2000; accepted 10 November 2000
Abstract We previously reported that the ES products from the plerocercoids of Spirometra erinaceieuropaei reduce nitric oxide synthase and chemokine gene expression in macrophages. In this study, we show that ES products suppressed tumor necrosis factor-a mRNA expression and tumor necrosis factor-a production in murine peritoneal macrophages stimulated with lipopolysaccharide or lipoteichoic acid in vitro. When macrophages from ES product-injected mice were stimulated with lipopolysaccharide in vitro, these cells produced smaller amounts of tumor necrosis factor-a compared with those taken from control mice. The suppressive effects of ES products were not restored by the treatment of indomethacin or anti-IL-10 antibody, and the ES products did not induce mRNA expression of secretory leukocyte protease inhibitor. Macrophages from C3H/HeJ mice, which have a single point mutation in the Toll-like receptor 4 gene, expressed tumor necrosis factor-a and IL-1a mRNA in the presence of lipopolysaccharide, but these expressions were less than those of macrophages from C3H/HeN. ES products signi®cantly suppressed tumor necrosis factor-a gene expression and tumor necrosis factor-a production in macrophages from C3H/HeN and C3H/HeJ mice stimulated with lipopolysaccharide. However, ES products had no effect on IL-1 mRNA expression. Our data suggest that the plerocercoids secrete the tumor necrosis factor-a inhibitory products to evade the host's immune system, and that tumor necrosis factor-a mRNA expression might be inhibited downstream from Toll-like receptor 4 in the lipopolysaccharide signaling pathway. q 2001 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. Keywords: Tumor necrosis factor-a; Macrophage; ES products; Lipopolysaccharide; Lipoteichoic acid; Spirometra erinaceieuropaei
1. Introduction Parasites need to avoid the host's immune system so that they can succeed in infecting the host. Although adult worms of Spirometra erinaceieuropaei usually parasitise the intestinal lumen of cats and dogs, the larval plerocercoids have low host-speci®city, and infection by plerocercoids causes `sparganosis' in humans. In many mammals, including rodents, plerocercoids, taken orally, separate their head from their body portions in the host's intestine and invade the host's peritoneal cavity. These plerocercoids may have various intestinal bacteria on their surfaces. The invading plerocercoids are covered with many bound in¯ammatory leukocytes, including macrophages. Although the macrophages and other leukocytes are activated with lipopolysaccharide (LPS) from gram-negative bacteria * Corresponding author. Tel.: 181-859-34-8028; fax: 181-859-34-8354. E-mail address: [email protected] (K. Miura).
and/or lipoteichoic acid (LTA) from gram-positive bacteria, the plerocercoids apparently survive unharmed. We presumed that plerocercoids produce a suppressive factor to inhibit in¯ammatory responses in the host, and we have shown that the ES products from the plerocercoids of S. erinaceieuropaei reduce the inducible isoform of nitric oxide synthase (iNOS) and the chemokines, JE and KC, in murine peritoneal macrophages (Fukumoto et al., 1997). Besides suppression of the expression of these genes, the plerocercoid produces plerocercoid growth factor/protease, which cleavages human IgG, while evading the host's immune defense system (Phares, 1996). Tumor necrosis factor (TNF)-a, a proin¯ammatory cytokine, protects against parasite infections, such as leishmaniasis (Tumang et al., 1994) and Chagas' disease (Lima et al., 1997). In the research reported here, we found that the ES products of plerocercoids suppressed TNF-a gene expression and production in mouse peritoneal macro-
0020-7519/01/$20.00 q 2001 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. PII: S 0020-751 9(00)00149-1
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phages in vitro and in vivo. Then, we examined whether TNF-a gene expression is suppressed by prostaglandin E2 (PGE2), IL-10 or secretory leukocyte protease inhibitor (SLPI); these suppressors are inducible in macrophages and repress macrophage functions autocrinely. Next, we examined how the ES products suppress TNF-a gene expression in macrophages stimulated with LPS or LTA. Based on the experiments with C3H/HeN and C3H/ HeJ mice macrophages, we wanted to know whether the suppression of TNF-a gene expression by ES products is related to Toll-like receptor 4 (TLR4). 2. Materials and methods 2.1. Plerocercoids of Spirometra erinaceieuropaei and their ES products Plerocercoids of S. erinaceieuropaei were collected from two species of snakes (Elarohe quadrivirgata and Rhabdophis tigrinus) in the eastern part of Shimane Prefecture, Japan. The plerocercoids from two or three snakes were stored in the s.c. tissue of a golden hamster for over 6 months. To obtain ES products, the plerocercoids were removed from the golden hamster aseptically and were washed three times in PBS with antibiotics. Fifty plerocercoids were incubated for 24 h in serum-free 50 ml Dulbecco's modi®ed Eagle's medium (DMEM) (GIBCO BRL), and, in turn, the incubation medium was dialyzed against distilled water and lyophilized. This sample was dissolved in DMEM and was adjusted to 50 mg/ml. Then it was sterilized using a 0.22 mm ®lter membrane for the in vitro experiments. The protein concentration was measured using a Bio-Rad protein assay kit (Bio-Rad Lab. Co.). We made separate lots of ES products from each golden hamster. Every experiment was repeated by using a different lot of ES independently and one representative experiment is shown. 2.2. Preparation and culture of peritoneal macrophages Male ICR mice (SLC, Inc.), C3H/HeN and C3H/HeJ mice (Clea Japan, Inc.), all 9±12 weeks of age, were utilized. These mice were kept in plastic cages on a 12 h light±dark cycle at 248C, and were fed pelleted murine food and offered water ad libitum. The mice were maintained in the Laboratory Animal Center, Faculty of Medicine, Tottori University, and received humane care in compliance with the guidelines stipulated under the National Institute of Health. Thioglycollate (Difco Laboratories)-elicited macrophages were harvested with 10 ml of ice-cold PBS on the fourth day after i.p. injection of 2 ml thioglycollate. The macrophages, in DMEM containing penicillin G (Banyu Pharmaceutical Co. Ltd.), streptomycin (Meiji Seika Ltd.) and 5% fetal calf serum (GIBCO BRL.), were plated in tissue culture dishes (Greiner), incubated at 378C in an
atmosphere of 10% CO2 and were left for more than 14 h before each experiment. 2.3. Preparation of total RNA and Northern hybridization analysis To obtain total RNA, 1 £ 10 7 macrophages were cultured in a 10 cm culture dish for the indicated times with the indicated stimuli. After treatment, total RNA was extracted by an ISOGEN Kit (Nippon Gene Ltd.). Northern hybridization analysis was performed as described elsewhere (Fukumoto et al., 1997). In brief, samples of total RNA (5.5 mg) were separated on an agarose gel and subsequently blotted onto a MagnaGraph Nylon transfer membrane (Micron Separations, Inc.). The blots were prehybridized and hybridized with [a- 32P]dCTP-radiolabeled cDNA plasmid probes prepared by a BcaBEST DNA Labeling kit (Takara Ltd.). Then, autoradiography was performed. Murine TNF-a and SLPI cDNA, consisting of a 433 and a 290 bp fragment, respectively, were cloned into the pGEM plasmid. The plasmids encoding the IL-1a and glyceraldehyde-3-phosphate dehydrogenate (GAPDH) were kindly provided by Professor Kenzo Sato (Tottori University) and Dr David Stern (Columbia University), respectively. The relative TNF-a, SLPI and IL-1a mRNA expressions were analyzed with a Molecular Imager, model GS-535 (Bio-Rad Lab. Co.), with reference to the expression of GAPDH. In the ®gures, the relative TNF-a/GAPDH, SLPI/GAPDH or IL-1a/GAPDH mRNA expression is presented as a percentage of the maximum. 2.4. ELISA for TNF-a The culture medium of 1 £ 10 7 macrophages in a 10 cm dish was harvested after incubation at the indicated times with the indicated stimuli in each ®gure legend, and stored at 2808C before measurement. The concentration of TNF-a was assayed using ELISA (Endogen, Inc.), following the manufacturer's protocol. In each experiment, the medium from four or six dishes were measured, and the means ^ standard errors are represented. 2.5. Time course and dose effects of ES products The macrophages were incubated for 1, 3 and 6 h with LPS (100 ng/ml) and/or ES products (5 mg/ml), and the total RNA were harvested. Next, the macrophages were incubated for 3 h with LPS (100 ng/ml) and/or different doses of ES products (0, 1, 5 and 10 mg/ml), and the total RNA and culture media were harvested. In the third series of experiments, the macrophages were stimulated with LPS (100 ng/ ml); 1 h after LPS addition, ES products (5 mg/ml) were poured into the culture dish. Total RNA and the culture medium were harvested 3 h after the addition of ES products.
K. Miura et al. / International Journal for Parasitology 31 (2000) 39±47
2.6. The effects of ES products in LTA-stimulated macrophages Total RNA and culture media were obtained from macrophages incubated with LTA (from Staphylococcus aureus; 10 mg/ml; Sigma Chemical Co.) in the presence or absence of ES products (5 mg/ml) for 3 h. 2.7. ES product injection in mice and macrophage collection To neutralize LPS contamination in ES products, polymyxin B (Sigma Chemical Co.) was mixed with ES products before injection. ICR mice were s.c. injected with ES products (10 or 100 mg/day) and polymyxin B (3 mg/day) dissolved in 0.1 ml of PBS into their backs for 4 days, while control mice were injected with the same volume of PBS including polymyxin B (3 mg/day) alone for 4 days. Then thioglycollate-elicited macrophages were obtained and were left for more than 14 h before stimulation of LPS. They were incubated for 3 h with LPS (100 ng/ml), and the total RNA and culture media were harvested.
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3. Results 3.1. Suppression of TNF-a mRNA expression and TNF-a release in macrophages by ES products The suppressive effects of ES products (5 mg/ml) on TNF-a mRNA expression were observed by Northern blot analysis in peritoneal macrophages stimulated with 100 ng/ ml LPS for 1, 3 and 6 h. ES products reduced the TNF-a gene expression by 14, 56 and 42%, respectively, compared with the expression in LPS-stimulated macrophages. Next, we examined dose effects of ES products. Exposure of LPS-activated macrophages to 5 or 10 mg/ml of ES products reduced the magnitude of TNF-a mRNA. Regarding the levels of GAPDH mRNA, none of the concentrations of ES products used had any effect on it (Fig. 1A,B). Not
2.8. The mechanisms of the suppression of TNF-a gene expression by ES products Macrophages were treated with 5 mg/ml ES products and/ or 3 mM indomethacin (Sigma Chemical Co.), to inhibit PGE2 synthesis, for 3 h before LPS (100 ng/ml) stimulation. Total RNA was obtained from macrophages 3 h after the addition of LPS. In the same way, macrophages were treated with ES products (5 mg/ml) and/or 10 mg/ml of rabbit antimouse IL-10 polyclonal antibody (Chemicon International, Inc.) for 3 h. After that, there were stimulated with LPS (100 ng/ml) for 3 h and total RNA was obtained. To observe the effects of ES products on SLPI gene expression, macrophages were incubated with LPS (100 ng/ml) and/or 5 mg/ml ES products for 3 h, and the total RNA was obtained. 2.9. Macrophages from C3H/HeN and C3H/HeJ mice were stimulated with LPS or LTA The macrophages from C3H/HeN and C3H/HeJ mice were preincubated with or without ES products (5 mg/ml) for 24 h. Next, they were stimulated with LPS (100 ng/ml) or LTA (10 mg/ml) for 3 h. Total RNA and the culture medium were harvested. The expressions of TNF-a, IL1a and GAPDH mRNA in macrophages stimulated with LPS or LTA were detected by Northern hybridization. TNF-a production in each culture medium was measured by ELISA.
Fig. 1. Dose effects of ES products on TNF-a mRNA expression and production in LPS-stimulated macrophages. The macrophages were incubated for 3 h with LPS (100 ng/ml) and/or ES products (0, 1, 5 and 10 mg/ ml), and the total RNA and culture media were harvested. (A) The expressions of TNF-a and GAPDH mRNA were detected by Northern hybridization. (B) The TNF-a and GAPDH mRNA levels were quanti®ed by a molecular imager. The relative TNF-a mRNA expression is shown. One representative experiment of two is shown. (C) TNF-a production in the culture medium was measured by ELISA. The data described here represent the means ^ standard errors (n 6) from one of three experiments. *P , 0:01, **P , 0:001 when TNF-a productions were compared by the Student's t-test.
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Fig. 2. The effects of ES products on TNF-a in LTA-stimulated macrophages. Total RNA and culture media were obtained from macrophages incubated with LTA (10 mg/ml) alone or with LTA (10 mg/ml) and ES products (5 mg/ml) for 3 h. (A) The expression of TNF-a and GAPDH mRNA were detected by Northern hybridization. (B) The TNF-a and GAPDH mRNA levels were quanti®ed by a molecular imager. The relative TNF-a mRNA expression is shown. One representative experiment of three is shown. (C) TNF-a production in the culture medium was measured by ELISA. The data described here represent the means ^ standard errors (n 6) from one of three experiments; where invisible, they fall within the bars. **P , 0:001 when TNF-a production was compared with LTA-stimulated macrophages by the Student's t-test. ND, not detected.
only the TNF-a mRNA expression, but also, TNF-a release in the cultured medium was measured by ELISA. ES products suppressed the TNF-a release in a dose dependent manner (Fig. 1C). However, a 1 mg/ml concentration of ES products had no signi®cant effect on TNF-a mRNA levels and TNF-a production. The heat-lability of ES products was tested by boiling for 5 min. The heat-treated ES or non-treated ES products (5 mg/ml) were added to 1.2 £ 10 6 macrophages in 3.5 cm culture dishes for 3 h with LPS (100 ng/ml), and the culture media were harvested for measurement of TNF-a. Although the original ES products suppressed the TNF-a release by 45% compared with the TNF-a release (3574 ^ 20 pg/ml) of LPS-stimulated macrophages, the boiled ES products did not suppress TNF-a production (3552 ^ 33 pg/ml). Many TNF-a suppressive agents are not as effective when they are added after the addition of LPS. However, ES products added 1 h after LPS stimulation signi®cantly
reduced TNF-a gene expression by 62% and TNF-a production by 58% (from 1730.9 ^ 69.8 to 729.1 ^ 19.7 pg/ml; P , 0:001). 3.2. ES products suppressed LTA-stimulated TNF-a production We observed that ES products reduce TNF-a production in macrophages stimulated by LTA (a component of the gram-positive bacterial cell wall), as well as LPS. Macrophages were incubated with LTA and ES products at the same time, and 3 h later, the total RNA and culture media were obtained. As with LPS stimulation, ES products significantly suppressed TNF-a mRNA expression and TNF-a production in LTA-stimulated macrophages (Fig. 2). 3.3. Injection of ES products in mice repressed the TNF-a production in macrophages ICR mice were injected with different doses of ES
Fig. 3. The effects of ES product injection in mice on TNF production of their macrophages in vitro. ICR mice were s.c. injected with polymyxin B alone (controls), or with polymyxin B and ES products (10 or 100 mg) for 4 days. Then, thioglycollate-elicited macrophages were obtained and left overnight (see Section 2). Total RNA and the incubation medium of macrophages were harvested 3 h after the addition of 100 ng/ml LPS in vitro. (A) The TNF-a and GAPDH mRNA levels were quanti®ed by a molecular imager. The relative TNF-a mRNA expression is shown. One representative experiment of two is shown. (B) TNF-a production in the culture medium was measured by ELISA. The data described here represent the means ^ standard errors (n 4) from one of two experiments. *P , 0:01, **P , 0:001 when TNF-a productions were compared by the Student's t-test. ND, not detected.
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Fig. 4. The effects of indomethacin and anti-IL-10 Ab on the suppression by ES products. Macrophages were treated with: (A,B), 5 mg/ml ES products and/or 3 mM indomethacin; or (C,D), 5 mg/ml ES products and/or 10 mg/ml of anti-IL-10 Ab, for 3 h before LPS (100 ng/ml) stimulation. Total RNA was obtained from macrophages 3 h after the addition of LPS. (A,C) The expression of TNF-a and GAPDH mRNA were detected by Northern hybridization. (B,D) The TNF-a and GAPDH mRNA levels were quanti®ed by a molecular imager. The relative TNF-a mRNA expression is shown. One representative experiment of two is shown.
products or the same volume of PBS into their backs s.c. for 4 days. Peritoneal macrophages were then obtained and stimulated with LPS in vitro. ES injection slightly suppressed TNF-a mRNA expression in LPS-stimulated macrophages (Fig. 3A), but the production of TNF-a in LPS-treated macrophages was signi®cantly suppressed in a dose dependent manner (Fig. 3B). 3.4. Effect of ES products was not mediated with PGE2 and IL-10 To determine whether the suppressive effect of ES products depends on PGE2 or IL-10 produced in macrophages, we used indomethacin, cyclooxygenase inhibitor and anti-IL-10 polyclonal antibody. Macrophages were treated with ES products and indomethacin (Fig. 4A,B), or with ES products and anti-IL-10 Ab (Fig. 4C,D) before LPS stimulation. As shown in Fig. 4, neither indomethacin nor
anti-IL-10 Ab could restore the suppression of TNF-a expression by ES products. 3.5. SLPI mRNA was not induced by ES products In order to determine whether SLPI was related to the suppression of TNF-a by the ES products, macrophages were treated with ES products and LPS for 3 h, and the total RNA was harvested. The SLPI mRNA expression was slightly repressed by ES products (Fig. 5). 3.6. ES products suppressed TNF-a production in macrophages from C3H/HeN and C3H/HeJ mice In the presence of LPS, macrophages from C3H/HeJ mice express 55% of TNF-a mRNA and 20% of IL-1a mRNA compared with those from C3H/HeN mice (Fig. 6A,B). TNF-a in the culture medium was measured by ELISA. C3H/HeJ macrophages produced only 6% of the TNF-a of C3H/HeN macrophages (Fig. 7). ES products signi®-
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C3H/HeJ mice. However, ES products had no effect on the levels of IL-1 mRNA in LPS-stimulated macrophages from both mice (Fig. 6A,B). In LTA-stimulated C3H/HeN macrophages, ES products also inhibited TNF-a mRNA expression, while the IL-1 mRNA level was not changed by ES products (Fig. 6C,D). In C3H/HeJ macrophages, LTA stimulation did not induce any TNF-a or IL-1 mRNA expression (Fig. 6C,D). 4. Discussion
Fig. 5. The effects of ES products on SLPI mRNA expression. Macrophages were incubated with LPS (100 ng/ml) and/or 5 mg/ml ES products for 3 h. Total RNA was obtained. The expression of SLPI mRNA was detected by Northern hybridization and quanti®ed by a molecular imager. The relative SLPI mRNA expression is shown. One representative experiment of two is shown.
cantly suppressed TNF-a mRNA expression and production in LPS-stimulated macrophages from both C3H/HeN and
We found that ES products suppressed TNF-a gene expression in LPS-stimulated macrophages in vitro experiments, in addition to iNOS and chemokines. When ES products were boiled for 5 min, their suppressive effects disappeared. Therefore, the suppressive factor in ES products may be a protein. Moreover, we have already partially puri®ed the suppressive factor from ES products. The suppressive factor was found in the void pool of gel ®ltration, in which protein bands were more than 94 kDa in SDS-PAGE (Fukumoto et al., 1998). Therefore, we decided to focus on the effective protein concentration of ES products (Fig. 1). From ES products, 5 mg/ml of protein suppressed TNF-a gene expression of 1 £ 10 7 macrophages
Fig. 6. The effects of ES products on TNF-a and IL-1 mRNA expression in LPS- or LTA-stimulated macrophages from C3H/HeN and C3H/HeJ mice. Macrophages were preincubated with or without ES products (5 mg/ml) for 24 h. Then, they were stimulated with LPS (100 ng/ml) or LTA (10 mg/ml). Total RNA and the culture medium were harvested from macrophages incubated for 3 h with: (A,B), LPS; or (C,D), LTA. The expressions of TNF-a, IL-1a and GAPDH mRNA in macrophages stimulated with LPS or LTA were detected by Northern hybridization. These gene expressions are shown in (A,C). The TNFa, IL-1a and GAPDH mRNA levels were quanti®ed by a molecular imager. The relative TNF-a and IL-1a mRNA expressions are shown in (B,D). One representative experiment of two is shown.
K. Miura et al. / International Journal for Parasitology 31 (2000) 39±47
Fig. 7. The effects of ES products on TNF-a production in LPS-stimulated macrophages from C3H/HeN and C3H/HeJ mice. Macrophages were preincubated with or without ES products (5 mg/ml) for 24 h. Then, they were stimulated with LPS (100 ng/ml). The culture medium was harvested from macrophages incubated for 3 h with LPS. TNF-a production in each culture medium was measured by ELISA. The data described here represent the means ^ standard errors (n 6) from one of two experiments; where invisible, they fall within the bars. **P , 0:001 when TNF-a productions were compared by the Student's t-test. ND, not detected
in vitro. One plerocercoid produces about 40 mg/day ES products, and at 378C, the suppressive effect did not diminish, even if the ES products were incubated for more than 24 h (data not shown). We next examined whether ES products suppressed TNF-a gene expression and TNF-a production in vivo. The suppressive effects were observed in peritoneal macrophages from ES product-injected mice in comparison with those in macrophages from control mice (Fig. 3). However, when we injected ES products into mice, it was dif®cult to judge whether the ES products suppressed the gene expression of the macrophages immediately or indirectly, such as changing the Th1/Th2 balance. Judging from our data, the suppressive factor in ES products might have some effect on TNF-a gene expression in the peritoneal macrophages in vivo. It has been reported that TNF-a expression in LPS-stimulated macrophages is inhibited by such agents as glucocorticoids (Sze¯er et al., 1989; Kutteh et al., 1991; Baumgartner et al., 1996; Chaudhri, 1997), IL-4 (Hart et al., 1989; Kambayashi et al., 1996; Levings and Schraderm, 1999), PGE2 (Kunkel et al., 1988; Strassmann et al., 1994; Barrios-Rodiles et al., 1999), IL-10 (Bogdan et al., 1992; Strassmann et al., 1994; Berg et al., 1995; Wang et al., 1995; Clarke et al., 1998), transforming growth factor-b (TGF-b) (Chantry et al., 1989; Bogdan et al., 1992) and SLPI (Jin et al., 1997, 1998a,b). We did not assume that the plerocercoids secrete the same suppressive factors as reported in mammals. Therefore, we examined whether ES products induce agents such as PGE2, IL-10, TGF-b and SLPI, which are inducible in macrophages and repress macrophage function autocrinely. There is no report that parasitic
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helminths excrete or secrete a macrophage suppressive factor. The LPS-stimulated TNF-a gene expression in Leishmania donovani-infected macrophages was suppressed, but the suppressive effect was completely restored with indomethacin, which inhibits PGE2 production by blocking cyclooxygenase activity (Descoteaux and Matlashewski, 1989). In the present investigation, the inhibitory effect of ES products was not affected by indomethacin (Fig. 4A,B); therefore, the mechanism of suppression by ES products was different from that of Leishmania infection. We found that IL-10 was not involved in the impairment of TNF-a gene expression by ES products, using anti-IL-10 antibody (Fig. 4C,D). Mouse SLPI cDNA was cloned (Jin et al., 1997) using a differential display to compare macrophage cell lines from two strains of mice (C3H/HeN and C3H/HeJ). Transfection with SLPI converts macrophages from LPS-sensitive to LPS-hyporesponsive, and TNF-a production in LPS-responsive cells was suppressed by transfecting with SLPI. In the present study, ES products did not upregulate SLPI mRNA expression in macrophages stimulated with LPS for 3 h (Fig. 5). TGF-b is one of the aforementioned candidates known as a suppressive factor. However, the TGF-b mRNA level did not change in macrophages co-cultivated with plerocercoids (unpublished data), while TNF-a gene expression was suppressed in those cells (Miura et al., 1998). Therefore, TGF-b may be not related to the suppressive effect of ES products. Furthermore, TGF-b and SLPI require more than 12 h to make a suppressive effect (Bogdan et al., 1992; Jin et al., 1998b). We examined TNF-a gene expression in macrophages after 3 h incubation, and ES products had a suppressive effect even when added 1 h after LPS stimulation. Therefore TGF-b and SLPI were not involved in the suppressive effect of ES products. Judging from these data, ES products did not induce the TNF-a suppressive factor in the macrophages mentioned above. In macrophages, LPS stimulation is mediated through a cell surface protein, CD14 (Wright et al., 1990). However, CD14 is a glycosylphosphatidylinositol-anchored protein without a transmembrane domain. Recent studies showed that one of the most important murine receptors of LPS is TLR4 (Chow et al., 1999; Hoshino et al., 1999; Takeuchi et al., 1999). TLR4 has been reported as a putative transmembrane receptor in mice, and it is responsible for the initiation of intracellular signal transduction after interaction with the LPS±CD14 or LTA±CD14 complex. In the experiment with TLR4 knock out mice (Takeuchi et al., 1999), it was revealed that not only LPS, but also LTA stimulation was mediated by TLR4. The C3H/HeJ mouse strain, characterized by hyporesponsiveness to LPS, has a single point mutation in the TLR 4 gene (Poltorak et al., 1998; Hoshino et al., 1999; Qureshi et al., 1999). The expression levels of both TNF-a and IL-1a mRNA in C3H/HeJ macrophages were lower than those in C3H/HeN macrophages in the presence of LPS (Fig. 6). Therefore, we hypothesize that LPS induced both TNF-a and IL-1 mRNA expression in the macrophages
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through TLR4. However, in LPS-stimulated macrophages from C3H/HeN mice, the TNF-a expression was suppressed with ES products, while the IL-1 gene expression was not changed. These data suggest that ES products inhibited TNF-a mRNA expression downstream from TLR4 in the LPS signal transduction. If CD14 or TLR4 were directly downregulated or blocked by ES products, IL-1 should also be suppressed. Although LPS-activated macrophages from C3H/HeJ mice express TNF-a and IL-1 mRNA, LTA-activated macrophages from C3H/HeJ did not express TNF-a or IL1 genes (Fig. 6). As for LPS receptors, macrophages have not only CD14±TLR4, but others (Hampton et al., 1991; Lynn and Golenbock, 1992; Ingalls and Golenbock, 1995; Ingalls et al., 1997; Medvedev et al., 1998). However, as for LTA-receptors, there has been no ef®cient receptor reported except TLR4. Therefore, TNF-a and IL-1 mRNA expression in LPS-activated macrophages from C3H/HeJ mice may be related to such receptors. Acknowledgements The authors are grateful to Professor Yuichi Ishibe, Department of Anesthesiology and Reanimatology, Faculty of Medicine, Tottori University, for his useful comments on the manuscript. This work was supported by a Grant-in-Aid for Scienti®c Research (numbers 09670258 and 11670241) from the Ministry of Science, Culture and Education, Japan. References Barrios-Rodiles, M., Tiraloche, G., Chadee, K., 1999. Lipopolysaccharide modulates cyclooxygenase-2 transcriptionally and posttranscriptionally in human macrophages independently from endogenous IL-1b and TNF-a. J. Immunol. 163, 963±9. Baumgartner, R.A., Deramo, V.A., Beaven, M.A., 1996. Constitutive and inducible mechanisms for synthesis and release of cytokines in immune cell lines. J. Immunol. 157, 4087±93. Berg, D.J., Kuhn, R., Rajewsky, K., Muller, W., Menon, S., Davidson, N., Grunig, G., Rennick, D., 1995. Interleukin-10 is a central regulator of the response to LPS in murine models of endotoxic shock and the Shwartzman reaction but not endotoxin tolerance. J. Clin. Invest. 96, 2339±47. Bogdan, C., Paik, J., Vodovotz, Y., Nathan, C., 1992. Contrasting mechanisms for suppression of macrophage cytokine release by transforming growth factor-b and interleukin-10. J. Biol. Chem. 267, 23301±8. Chantry, D., Turner, M., Abney, E., Feldmann, M., 1989. Modulation of cytokine production by transforming growth factor-b. J. Immunol. 142, 4295±300. Chaudhri, G., 1997. Differential regulation of biosynthesis of cell surface and secreted TNF-a in LPS-stimulated murine macrophages. J. Leukoc. Biol. 62, 249±57. Chow, J.C., Young, D.W., Golenbock, D.T., Christ, W.J., Gusovsky, F., 1999. Toll-like receptor-4 mediates lipopolysaccharide-induced signal transduction. J. Biol. Chem. 274, 10689±92. Clarke, C.J.P., Hales, A., Hunt, A., Foxwell, B.M.J., 1998. IL-10-mediated suppression of TNF-a production is independent of its ability to inhibit NF-kB activity. Eur. J. Immunol. 28, 1719±26. Descoteaux, A., Matlashewski, G., 1989. c-fos and tumor necrosis factor
gene expression in Leishmania donovani-infected macrophages. Mol. Cell Biol. 9, 5223±7. Fukumoto, S., Hirai, K., Tanihata, T., Ohmori, Y., Stuehr, D.J., Hamilton, T.A., 1997. Excretory/secretory products from plerocercoids of Spirometra erinacei reduce iNOS and chemokine mRNA levels in peritoneal macrophages stimulated with cytokines and/or LPS. Parasite Immunol. 19, 325±32. Fukumoto, S., Miura, K., Tanihata, T., Wang, H., Tademoto, S., Maejima, J., Hirai, K., 1998. Excretory/secretory products from Spirometra erinaceieuropaei reduce iNOS and chemokine mRNA levels in murine macrophages stimulated with cytokines and/or LPS. In: Tada, I., Kojima, S., Tsuji, M. (Eds.), Ninth International Congress of Parasitology, Monduzzi Editore, Bologna, pp. 545±50. Hampton, R.Y., Golenbock, D.T., Penman, M., Krieger, M., Raetz, C.R.H., 1991. Recognition and plasma clearance of endotoxin by scavenger receptors. Nature 352, 342±4. Hart, P.H., Vitti, G.F., Burgess, D.R., Whitty, G.A., Piccoli, D.S., Hamilton, J.A., 1989. Potential antiin¯ammatory effects of interleukin 4: suppression of human monocyte tumor necrosis factor a, interleukin 1, and prostaglandin E2. Proc. Natl. Acad. Sci. USA 86, 3803±7. Hoshino, K., Takeuchi, O., Kawai, T., Sanjo, H., Ogawa, T., Takeda, Y., Takeda, K., Akira, S., 1999. Toll-like receptor 4 (TLR4)-de®cient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product. J. Immunol. 162, 3749±52. Ingalls, R.R., Golenbock, D.T., 1995. CD11c/CD18, a transmembrane signaling receptor for lipopolysaccharide. J. Exp. Med. 181, 1473±9. Ingalls, R.R., Arnaout, M.A., Golenbock, D.T., 1997. Outside±in signaling by lipopolysaccharide through a tailless integrin. J. Immunol. 159, 433± 8. Jin, F.Y., Nathan, C., Radzioch, D., Ding, A., 1997. Secretory leukocyte protease inhibitor: a macrophage product induced by and antagonistic to bacterial lipopolysaccharide. Cell 88, 417±26. Jin, F., Nathan, C.F., Radzioch, D., Ding, A., 1998a. Lipopolysacchariderelated stimuli induce expression of the secretory leukocyte protease inhibitor, a macrophage-derived lipopolysaccharide inhibitor. Infect. Immun. 66, 2447±52. Jin, F., Nathan, C., Ding, A., 1998b. Identi®cation of genes involved in innate responsiveness to bacterial products by differential display. Methods 16, 396±406. Kambayashi, T., Jacob, C.O., Strassmann, G., 1996. IL-4 and IL-13 modulate IL-10 release in endotoxin-stimulated murine peritoneal mononuclear phagocytes. Cell Immunol. 171, 153±8. Kunkel, S.L., Spengler, M., May, M.A., Spengler, R., Larrick, J., Remick, D., 1988. Prostaglandin E2 regulates macrophage-derived tumor necrosis factor gene expression. J. Biol. Chem. 263, 5380±4. Kutteh, W.H., Rainey, W.E., Carr, B.R., 1991. Glucocorticoids inhibit lipopolysaccharide-induced production of tumor necrosis factor-a by human fetal Kupffer cells. J. Clin. Endocrinol. Metab. 73, 296±301. Levings, M.K., Schraderm, J.W., 1999. IL-4 inhibits the production of TNF-a and IL-12 by STAT6-dependent and -independent mechanisms. J. Immunol. 162, 5224±9. Lima, E.C.S., Garcia, I., Vicentelli, M.H., Vassalli, P., Minoprio, P., 1997. Evidence for a protective role of tumor necrosis factor in the acute phase of Trypanosoma cruzi infection in mice. Infect. Immun. 65, 457±65. Lynn, W.A., Golenbock, D.T., 1992. Lipopolysaccharide antagonists. Immunol. Today 13, 271±6. Medvedev, A.E., Flo, T., Ingalls, R.R., Golenbock, D.T., Teti, G., Vogel, S.N., Espevik, T., 1998. Involvement of CD14 and complement receptors CR3 and CR4 in nuclear factor-kB activation and TNF production induced by lipopolysaccharide and group B streptococcal cell walls. J. Immunol. 160, 4535±42. Miura, K., Tanihata, T., Fukumoto, S., Wang, H., Hirai, K., 1998. Plerocercoids of Spirometra erinaceieuropaei reduce gene expression of tumor necrosis factor-a in mice. In: Tada, I., Kojima, S., Tsuji, M. (Eds.). Ninth International Congress of Parasitology, Monduzzi Editore, Bologna, pp. 1241±5.
K. Miura et al. / International Journal for Parasitology 31 (2000) 39±47 Phares, K., 1996. An unusual host±parasite relationship: the growth hormone-like factor from plerocercoids of spirometrid tapeworms. Int. J. Parasitol. 26, 575±88. Poltorak, A., He, X., Smirnova, I., Liu, M.Y., Huffel, C.V., Du, X., Birdwell, D., Alejos, E., Silva, M., Galanos, C., Freudenberg, M., Ricciardi Castagnoli, P., Layton, B., Beutler, B., 1998. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282, 2085±8. Qureshi, S.T., Lariviere, L., Leveque, G., Clermont, S., Moore, K.J., Gros, P., Malo, D., 1999. Endotoxin-tolerant mice have mutations in Toll-like receptor 4 (Tlr4). J. Exp. Med. 189, 615±25. Strassmann, G., Patil-Koota, V., Finkelman, F., Fong, M., Kambayashi, T., 1994. Evidence for the involvement of interleukin 10 in the differential deactivation of murine peritoneal macrophages by prostaglandin E2. J. Exp. Med. 180, 2365±70. Sze¯er, S.J., Norton, C.E., Ball, B., Gross, J.M., Aida, Y., Pabst, M.J., 1989. IFN-g and LPS overcome glucocorticoid inhibition of priming for
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superoxide release in human monocytes. Evidence that secretion of IL-1 and tumor necrosis factor-a is not essential for monocyte priming. J. Immunol. 142, 3985±92. Takeuchi, O., Hoshino, K., Kawai, T., Sanjo, H., Takada, H., Ogawa, T., Takeda, K., Akira, S., 1999. Differential roles of TLR2 and TLR4 in recognition of gram-negative and gram-positive bacterial cell wall components. Immunity 11, 443±51. Tumang, M.C., Keogh, C., Moldawer, L.L., Helfgott, D.C., Teitelbaum, R., Hariprashad, J., Murray, H.W., 1994. Role and effect of TNF-a in experimental visceral leishmaniasis. J. Immunol. 153, 768±75. Wang, P., Wu, P., Siegel, M.I., Egan, R.W., Billah, M.M., 1995. Interleukin (IL)-10 inhibits nuclear factor kB (NF-kB) activation in human monocytes. IL-10 and IL-4 suppress cytokine synthesis by different mechanisms. J. Biol. Chem. 270, 9558±63. Wright, S.D., Ramos, R.A., Tobias, P.S., Ulevitch, R.J., Mathison, J.C., 1990. CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein. Science 249, 1431±43.
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Excretory/secretory products from plerocercoids of Spirometra erinaceieuropaei suppress gene expressions and production of tumor necrosis factor-a in murine macrophages stimulated with lipopolysaccharide or lipoteichoic acid K. Miura*, S. Fukumoto, P. Dirgahayu, K. Hirai Department of Medical Zoology, Faculty of Medicine, Tottori University, 86 Nishi-Cho, Yonago 683-8503, Japan Received 5 September 2000; received in revised form 10 November 2000; accepted 10 November 2000
Abstract We previously reported that the ES products from the plerocercoids of Spirometra erinaceieuropaei reduce nitric oxide synthase and chemokine gene expression in macrophages. In this study, we show that ES products suppressed tumor necrosis factor-a mRNA expression and tumor necrosis factor-a production in murine peritoneal macrophages stimulated with lipopolysaccharide or lipoteichoic acid in vitro. When macrophages from ES product-injected mice were stimulated with lipopolysaccharide in vitro, these cells produced smaller amounts of tumor necrosis factor-a compared with those taken from control mice. The suppressive effects of ES products were not restored by the treatment of indomethacin or anti-IL-10 antibody, and the ES products did not induce mRNA expression of secretory leukocyte protease inhibitor. Macrophages from C3H/HeJ mice, which have a single point mutation in the Toll-like receptor 4 gene, expressed tumor necrosis factor-a and IL-1a mRNA in the presence of lipopolysaccharide, but these expressions were less than those of macrophages from C3H/HeN. ES products signi®cantly suppressed tumor necrosis factor-a gene expression and tumor necrosis factor-a production in macrophages from C3H/HeN and C3H/HeJ mice stimulated with lipopolysaccharide. However, ES products had no effect on IL-1 mRNA expression. Our data suggest that the plerocercoids secrete the tumor necrosis factor-a inhibitory products to evade the host's immune system, and that tumor necrosis factor-a mRNA expression might be inhibited downstream from Toll-like receptor 4 in the lipopolysaccharide signaling pathway. q 2001 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. Keywords: Tumor necrosis factor-a; Macrophage; ES products; Lipopolysaccharide; Lipoteichoic acid; Spirometra erinaceieuropaei
1. Introduction Parasites need to avoid the host's immune system so that they can succeed in infecting the host. Although adult worms of Spirometra erinaceieuropaei usually parasitise the intestinal lumen of cats and dogs, the larval plerocercoids have low host-speci®city, and infection by plerocercoids causes `sparganosis' in humans. In many mammals, including rodents, plerocercoids, taken orally, separate their head from their body portions in the host's intestine and invade the host's peritoneal cavity. These plerocercoids may have various intestinal bacteria on their surfaces. The invading plerocercoids are covered with many bound in¯ammatory leukocytes, including macrophages. Although the macrophages and other leukocytes are activated with lipopolysaccharide (LPS) from gram-negative bacteria * Corresponding author. Tel.: 181-859-34-8028; fax: 181-859-34-8354. E-mail address: [email protected] (K. Miura).
and/or lipoteichoic acid (LTA) from gram-positive bacteria, the plerocercoids apparently survive unharmed. We presumed that plerocercoids produce a suppressive factor to inhibit in¯ammatory responses in the host, and we have shown that the ES products from the plerocercoids of S. erinaceieuropaei reduce the inducible isoform of nitric oxide synthase (iNOS) and the chemokines, JE and KC, in murine peritoneal macrophages (Fukumoto et al., 1997). Besides suppression of the expression of these genes, the plerocercoid produces plerocercoid growth factor/protease, which cleavages human IgG, while evading the host's immune defense system (Phares, 1996). Tumor necrosis factor (TNF)-a, a proin¯ammatory cytokine, protects against parasite infections, such as leishmaniasis (Tumang et al., 1994) and Chagas' disease (Lima et al., 1997). In the research reported here, we found that the ES products of plerocercoids suppressed TNF-a gene expression and production in mouse peritoneal macro-
0020-7519/01/$20.00 q 2001 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. PII: S 0020-751 9(00)00149-1
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phages in vitro and in vivo. Then, we examined whether TNF-a gene expression is suppressed by prostaglandin E2 (PGE2), IL-10 or secretory leukocyte protease inhibitor (SLPI); these suppressors are inducible in macrophages and repress macrophage functions autocrinely. Next, we examined how the ES products suppress TNF-a gene expression in macrophages stimulated with LPS or LTA. Based on the experiments with C3H/HeN and C3H/ HeJ mice macrophages, we wanted to know whether the suppression of TNF-a gene expression by ES products is related to Toll-like receptor 4 (TLR4). 2. Materials and methods 2.1. Plerocercoids of Spirometra erinaceieuropaei and their ES products Plerocercoids of S. erinaceieuropaei were collected from two species of snakes (Elarohe quadrivirgata and Rhabdophis tigrinus) in the eastern part of Shimane Prefecture, Japan. The plerocercoids from two or three snakes were stored in the s.c. tissue of a golden hamster for over 6 months. To obtain ES products, the plerocercoids were removed from the golden hamster aseptically and were washed three times in PBS with antibiotics. Fifty plerocercoids were incubated for 24 h in serum-free 50 ml Dulbecco's modi®ed Eagle's medium (DMEM) (GIBCO BRL), and, in turn, the incubation medium was dialyzed against distilled water and lyophilized. This sample was dissolved in DMEM and was adjusted to 50 mg/ml. Then it was sterilized using a 0.22 mm ®lter membrane for the in vitro experiments. The protein concentration was measured using a Bio-Rad protein assay kit (Bio-Rad Lab. Co.). We made separate lots of ES products from each golden hamster. Every experiment was repeated by using a different lot of ES independently and one representative experiment is shown. 2.2. Preparation and culture of peritoneal macrophages Male ICR mice (SLC, Inc.), C3H/HeN and C3H/HeJ mice (Clea Japan, Inc.), all 9±12 weeks of age, were utilized. These mice were kept in plastic cages on a 12 h light±dark cycle at 248C, and were fed pelleted murine food and offered water ad libitum. The mice were maintained in the Laboratory Animal Center, Faculty of Medicine, Tottori University, and received humane care in compliance with the guidelines stipulated under the National Institute of Health. Thioglycollate (Difco Laboratories)-elicited macrophages were harvested with 10 ml of ice-cold PBS on the fourth day after i.p. injection of 2 ml thioglycollate. The macrophages, in DMEM containing penicillin G (Banyu Pharmaceutical Co. Ltd.), streptomycin (Meiji Seika Ltd.) and 5% fetal calf serum (GIBCO BRL.), were plated in tissue culture dishes (Greiner), incubated at 378C in an
atmosphere of 10% CO2 and were left for more than 14 h before each experiment. 2.3. Preparation of total RNA and Northern hybridization analysis To obtain total RNA, 1 £ 10 7 macrophages were cultured in a 10 cm culture dish for the indicated times with the indicated stimuli. After treatment, total RNA was extracted by an ISOGEN Kit (Nippon Gene Ltd.). Northern hybridization analysis was performed as described elsewhere (Fukumoto et al., 1997). In brief, samples of total RNA (5.5 mg) were separated on an agarose gel and subsequently blotted onto a MagnaGraph Nylon transfer membrane (Micron Separations, Inc.). The blots were prehybridized and hybridized with [a- 32P]dCTP-radiolabeled cDNA plasmid probes prepared by a BcaBEST DNA Labeling kit (Takara Ltd.). Then, autoradiography was performed. Murine TNF-a and SLPI cDNA, consisting of a 433 and a 290 bp fragment, respectively, were cloned into the pGEM plasmid. The plasmids encoding the IL-1a and glyceraldehyde-3-phosphate dehydrogenate (GAPDH) were kindly provided by Professor Kenzo Sato (Tottori University) and Dr David Stern (Columbia University), respectively. The relative TNF-a, SLPI and IL-1a mRNA expressions were analyzed with a Molecular Imager, model GS-535 (Bio-Rad Lab. Co.), with reference to the expression of GAPDH. In the ®gures, the relative TNF-a/GAPDH, SLPI/GAPDH or IL-1a/GAPDH mRNA expression is presented as a percentage of the maximum. 2.4. ELISA for TNF-a The culture medium of 1 £ 10 7 macrophages in a 10 cm dish was harvested after incubation at the indicated times with the indicated stimuli in each ®gure legend, and stored at 2808C before measurement. The concentration of TNF-a was assayed using ELISA (Endogen, Inc.), following the manufacturer's protocol. In each experiment, the medium from four or six dishes were measured, and the means ^ standard errors are represented. 2.5. Time course and dose effects of ES products The macrophages were incubated for 1, 3 and 6 h with LPS (100 ng/ml) and/or ES products (5 mg/ml), and the total RNA were harvested. Next, the macrophages were incubated for 3 h with LPS (100 ng/ml) and/or different doses of ES products (0, 1, 5 and 10 mg/ml), and the total RNA and culture media were harvested. In the third series of experiments, the macrophages were stimulated with LPS (100 ng/ ml); 1 h after LPS addition, ES products (5 mg/ml) were poured into the culture dish. Total RNA and the culture medium were harvested 3 h after the addition of ES products.
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2.6. The effects of ES products in LTA-stimulated macrophages Total RNA and culture media were obtained from macrophages incubated with LTA (from Staphylococcus aureus; 10 mg/ml; Sigma Chemical Co.) in the presence or absence of ES products (5 mg/ml) for 3 h. 2.7. ES product injection in mice and macrophage collection To neutralize LPS contamination in ES products, polymyxin B (Sigma Chemical Co.) was mixed with ES products before injection. ICR mice were s.c. injected with ES products (10 or 100 mg/day) and polymyxin B (3 mg/day) dissolved in 0.1 ml of PBS into their backs for 4 days, while control mice were injected with the same volume of PBS including polymyxin B (3 mg/day) alone for 4 days. Then thioglycollate-elicited macrophages were obtained and were left for more than 14 h before stimulation of LPS. They were incubated for 3 h with LPS (100 ng/ml), and the total RNA and culture media were harvested.
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3. Results 3.1. Suppression of TNF-a mRNA expression and TNF-a release in macrophages by ES products The suppressive effects of ES products (5 mg/ml) on TNF-a mRNA expression were observed by Northern blot analysis in peritoneal macrophages stimulated with 100 ng/ ml LPS for 1, 3 and 6 h. ES products reduced the TNF-a gene expression by 14, 56 and 42%, respectively, compared with the expression in LPS-stimulated macrophages. Next, we examined dose effects of ES products. Exposure of LPS-activated macrophages to 5 or 10 mg/ml of ES products reduced the magnitude of TNF-a mRNA. Regarding the levels of GAPDH mRNA, none of the concentrations of ES products used had any effect on it (Fig. 1A,B). Not
2.8. The mechanisms of the suppression of TNF-a gene expression by ES products Macrophages were treated with 5 mg/ml ES products and/ or 3 mM indomethacin (Sigma Chemical Co.), to inhibit PGE2 synthesis, for 3 h before LPS (100 ng/ml) stimulation. Total RNA was obtained from macrophages 3 h after the addition of LPS. In the same way, macrophages were treated with ES products (5 mg/ml) and/or 10 mg/ml of rabbit antimouse IL-10 polyclonal antibody (Chemicon International, Inc.) for 3 h. After that, there were stimulated with LPS (100 ng/ml) for 3 h and total RNA was obtained. To observe the effects of ES products on SLPI gene expression, macrophages were incubated with LPS (100 ng/ml) and/or 5 mg/ml ES products for 3 h, and the total RNA was obtained. 2.9. Macrophages from C3H/HeN and C3H/HeJ mice were stimulated with LPS or LTA The macrophages from C3H/HeN and C3H/HeJ mice were preincubated with or without ES products (5 mg/ml) for 24 h. Next, they were stimulated with LPS (100 ng/ml) or LTA (10 mg/ml) for 3 h. Total RNA and the culture medium were harvested. The expressions of TNF-a, IL1a and GAPDH mRNA in macrophages stimulated with LPS or LTA were detected by Northern hybridization. TNF-a production in each culture medium was measured by ELISA.
Fig. 1. Dose effects of ES products on TNF-a mRNA expression and production in LPS-stimulated macrophages. The macrophages were incubated for 3 h with LPS (100 ng/ml) and/or ES products (0, 1, 5 and 10 mg/ ml), and the total RNA and culture media were harvested. (A) The expressions of TNF-a and GAPDH mRNA were detected by Northern hybridization. (B) The TNF-a and GAPDH mRNA levels were quanti®ed by a molecular imager. The relative TNF-a mRNA expression is shown. One representative experiment of two is shown. (C) TNF-a production in the culture medium was measured by ELISA. The data described here represent the means ^ standard errors (n 6) from one of three experiments. *P , 0:01, **P , 0:001 when TNF-a productions were compared by the Student's t-test.
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Fig. 2. The effects of ES products on TNF-a in LTA-stimulated macrophages. Total RNA and culture media were obtained from macrophages incubated with LTA (10 mg/ml) alone or with LTA (10 mg/ml) and ES products (5 mg/ml) for 3 h. (A) The expression of TNF-a and GAPDH mRNA were detected by Northern hybridization. (B) The TNF-a and GAPDH mRNA levels were quanti®ed by a molecular imager. The relative TNF-a mRNA expression is shown. One representative experiment of three is shown. (C) TNF-a production in the culture medium was measured by ELISA. The data described here represent the means ^ standard errors (n 6) from one of three experiments; where invisible, they fall within the bars. **P , 0:001 when TNF-a production was compared with LTA-stimulated macrophages by the Student's t-test. ND, not detected.
only the TNF-a mRNA expression, but also, TNF-a release in the cultured medium was measured by ELISA. ES products suppressed the TNF-a release in a dose dependent manner (Fig. 1C). However, a 1 mg/ml concentration of ES products had no signi®cant effect on TNF-a mRNA levels and TNF-a production. The heat-lability of ES products was tested by boiling for 5 min. The heat-treated ES or non-treated ES products (5 mg/ml) were added to 1.2 £ 10 6 macrophages in 3.5 cm culture dishes for 3 h with LPS (100 ng/ml), and the culture media were harvested for measurement of TNF-a. Although the original ES products suppressed the TNF-a release by 45% compared with the TNF-a release (3574 ^ 20 pg/ml) of LPS-stimulated macrophages, the boiled ES products did not suppress TNF-a production (3552 ^ 33 pg/ml). Many TNF-a suppressive agents are not as effective when they are added after the addition of LPS. However, ES products added 1 h after LPS stimulation signi®cantly
reduced TNF-a gene expression by 62% and TNF-a production by 58% (from 1730.9 ^ 69.8 to 729.1 ^ 19.7 pg/ml; P , 0:001). 3.2. ES products suppressed LTA-stimulated TNF-a production We observed that ES products reduce TNF-a production in macrophages stimulated by LTA (a component of the gram-positive bacterial cell wall), as well as LPS. Macrophages were incubated with LTA and ES products at the same time, and 3 h later, the total RNA and culture media were obtained. As with LPS stimulation, ES products significantly suppressed TNF-a mRNA expression and TNF-a production in LTA-stimulated macrophages (Fig. 2). 3.3. Injection of ES products in mice repressed the TNF-a production in macrophages ICR mice were injected with different doses of ES
Fig. 3. The effects of ES product injection in mice on TNF production of their macrophages in vitro. ICR mice were s.c. injected with polymyxin B alone (controls), or with polymyxin B and ES products (10 or 100 mg) for 4 days. Then, thioglycollate-elicited macrophages were obtained and left overnight (see Section 2). Total RNA and the incubation medium of macrophages were harvested 3 h after the addition of 100 ng/ml LPS in vitro. (A) The TNF-a and GAPDH mRNA levels were quanti®ed by a molecular imager. The relative TNF-a mRNA expression is shown. One representative experiment of two is shown. (B) TNF-a production in the culture medium was measured by ELISA. The data described here represent the means ^ standard errors (n 4) from one of two experiments. *P , 0:01, **P , 0:001 when TNF-a productions were compared by the Student's t-test. ND, not detected.
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Fig. 4. The effects of indomethacin and anti-IL-10 Ab on the suppression by ES products. Macrophages were treated with: (A,B), 5 mg/ml ES products and/or 3 mM indomethacin; or (C,D), 5 mg/ml ES products and/or 10 mg/ml of anti-IL-10 Ab, for 3 h before LPS (100 ng/ml) stimulation. Total RNA was obtained from macrophages 3 h after the addition of LPS. (A,C) The expression of TNF-a and GAPDH mRNA were detected by Northern hybridization. (B,D) The TNF-a and GAPDH mRNA levels were quanti®ed by a molecular imager. The relative TNF-a mRNA expression is shown. One representative experiment of two is shown.
products or the same volume of PBS into their backs s.c. for 4 days. Peritoneal macrophages were then obtained and stimulated with LPS in vitro. ES injection slightly suppressed TNF-a mRNA expression in LPS-stimulated macrophages (Fig. 3A), but the production of TNF-a in LPS-treated macrophages was signi®cantly suppressed in a dose dependent manner (Fig. 3B). 3.4. Effect of ES products was not mediated with PGE2 and IL-10 To determine whether the suppressive effect of ES products depends on PGE2 or IL-10 produced in macrophages, we used indomethacin, cyclooxygenase inhibitor and anti-IL-10 polyclonal antibody. Macrophages were treated with ES products and indomethacin (Fig. 4A,B), or with ES products and anti-IL-10 Ab (Fig. 4C,D) before LPS stimulation. As shown in Fig. 4, neither indomethacin nor
anti-IL-10 Ab could restore the suppression of TNF-a expression by ES products. 3.5. SLPI mRNA was not induced by ES products In order to determine whether SLPI was related to the suppression of TNF-a by the ES products, macrophages were treated with ES products and LPS for 3 h, and the total RNA was harvested. The SLPI mRNA expression was slightly repressed by ES products (Fig. 5). 3.6. ES products suppressed TNF-a production in macrophages from C3H/HeN and C3H/HeJ mice In the presence of LPS, macrophages from C3H/HeJ mice express 55% of TNF-a mRNA and 20% of IL-1a mRNA compared with those from C3H/HeN mice (Fig. 6A,B). TNF-a in the culture medium was measured by ELISA. C3H/HeJ macrophages produced only 6% of the TNF-a of C3H/HeN macrophages (Fig. 7). ES products signi®-
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C3H/HeJ mice. However, ES products had no effect on the levels of IL-1 mRNA in LPS-stimulated macrophages from both mice (Fig. 6A,B). In LTA-stimulated C3H/HeN macrophages, ES products also inhibited TNF-a mRNA expression, while the IL-1 mRNA level was not changed by ES products (Fig. 6C,D). In C3H/HeJ macrophages, LTA stimulation did not induce any TNF-a or IL-1 mRNA expression (Fig. 6C,D). 4. Discussion
Fig. 5. The effects of ES products on SLPI mRNA expression. Macrophages were incubated with LPS (100 ng/ml) and/or 5 mg/ml ES products for 3 h. Total RNA was obtained. The expression of SLPI mRNA was detected by Northern hybridization and quanti®ed by a molecular imager. The relative SLPI mRNA expression is shown. One representative experiment of two is shown.
cantly suppressed TNF-a mRNA expression and production in LPS-stimulated macrophages from both C3H/HeN and
We found that ES products suppressed TNF-a gene expression in LPS-stimulated macrophages in vitro experiments, in addition to iNOS and chemokines. When ES products were boiled for 5 min, their suppressive effects disappeared. Therefore, the suppressive factor in ES products may be a protein. Moreover, we have already partially puri®ed the suppressive factor from ES products. The suppressive factor was found in the void pool of gel ®ltration, in which protein bands were more than 94 kDa in SDS-PAGE (Fukumoto et al., 1998). Therefore, we decided to focus on the effective protein concentration of ES products (Fig. 1). From ES products, 5 mg/ml of protein suppressed TNF-a gene expression of 1 £ 10 7 macrophages
Fig. 6. The effects of ES products on TNF-a and IL-1 mRNA expression in LPS- or LTA-stimulated macrophages from C3H/HeN and C3H/HeJ mice. Macrophages were preincubated with or without ES products (5 mg/ml) for 24 h. Then, they were stimulated with LPS (100 ng/ml) or LTA (10 mg/ml). Total RNA and the culture medium were harvested from macrophages incubated for 3 h with: (A,B), LPS; or (C,D), LTA. The expressions of TNF-a, IL-1a and GAPDH mRNA in macrophages stimulated with LPS or LTA were detected by Northern hybridization. These gene expressions are shown in (A,C). The TNFa, IL-1a and GAPDH mRNA levels were quanti®ed by a molecular imager. The relative TNF-a and IL-1a mRNA expressions are shown in (B,D). One representative experiment of two is shown.
K. Miura et al. / International Journal for Parasitology 31 (2000) 39±47
Fig. 7. The effects of ES products on TNF-a production in LPS-stimulated macrophages from C3H/HeN and C3H/HeJ mice. Macrophages were preincubated with or without ES products (5 mg/ml) for 24 h. Then, they were stimulated with LPS (100 ng/ml). The culture medium was harvested from macrophages incubated for 3 h with LPS. TNF-a production in each culture medium was measured by ELISA. The data described here represent the means ^ standard errors (n 6) from one of two experiments; where invisible, they fall within the bars. **P , 0:001 when TNF-a productions were compared by the Student's t-test. ND, not detected
in vitro. One plerocercoid produces about 40 mg/day ES products, and at 378C, the suppressive effect did not diminish, even if the ES products were incubated for more than 24 h (data not shown). We next examined whether ES products suppressed TNF-a gene expression and TNF-a production in vivo. The suppressive effects were observed in peritoneal macrophages from ES product-injected mice in comparison with those in macrophages from control mice (Fig. 3). However, when we injected ES products into mice, it was dif®cult to judge whether the ES products suppressed the gene expression of the macrophages immediately or indirectly, such as changing the Th1/Th2 balance. Judging from our data, the suppressive factor in ES products might have some effect on TNF-a gene expression in the peritoneal macrophages in vivo. It has been reported that TNF-a expression in LPS-stimulated macrophages is inhibited by such agents as glucocorticoids (Sze¯er et al., 1989; Kutteh et al., 1991; Baumgartner et al., 1996; Chaudhri, 1997), IL-4 (Hart et al., 1989; Kambayashi et al., 1996; Levings and Schraderm, 1999), PGE2 (Kunkel et al., 1988; Strassmann et al., 1994; Barrios-Rodiles et al., 1999), IL-10 (Bogdan et al., 1992; Strassmann et al., 1994; Berg et al., 1995; Wang et al., 1995; Clarke et al., 1998), transforming growth factor-b (TGF-b) (Chantry et al., 1989; Bogdan et al., 1992) and SLPI (Jin et al., 1997, 1998a,b). We did not assume that the plerocercoids secrete the same suppressive factors as reported in mammals. Therefore, we examined whether ES products induce agents such as PGE2, IL-10, TGF-b and SLPI, which are inducible in macrophages and repress macrophage function autocrinely. There is no report that parasitic
45
helminths excrete or secrete a macrophage suppressive factor. The LPS-stimulated TNF-a gene expression in Leishmania donovani-infected macrophages was suppressed, but the suppressive effect was completely restored with indomethacin, which inhibits PGE2 production by blocking cyclooxygenase activity (Descoteaux and Matlashewski, 1989). In the present investigation, the inhibitory effect of ES products was not affected by indomethacin (Fig. 4A,B); therefore, the mechanism of suppression by ES products was different from that of Leishmania infection. We found that IL-10 was not involved in the impairment of TNF-a gene expression by ES products, using anti-IL-10 antibody (Fig. 4C,D). Mouse SLPI cDNA was cloned (Jin et al., 1997) using a differential display to compare macrophage cell lines from two strains of mice (C3H/HeN and C3H/HeJ). Transfection with SLPI converts macrophages from LPS-sensitive to LPS-hyporesponsive, and TNF-a production in LPS-responsive cells was suppressed by transfecting with SLPI. In the present study, ES products did not upregulate SLPI mRNA expression in macrophages stimulated with LPS for 3 h (Fig. 5). TGF-b is one of the aforementioned candidates known as a suppressive factor. However, the TGF-b mRNA level did not change in macrophages co-cultivated with plerocercoids (unpublished data), while TNF-a gene expression was suppressed in those cells (Miura et al., 1998). Therefore, TGF-b may be not related to the suppressive effect of ES products. Furthermore, TGF-b and SLPI require more than 12 h to make a suppressive effect (Bogdan et al., 1992; Jin et al., 1998b). We examined TNF-a gene expression in macrophages after 3 h incubation, and ES products had a suppressive effect even when added 1 h after LPS stimulation. Therefore TGF-b and SLPI were not involved in the suppressive effect of ES products. Judging from these data, ES products did not induce the TNF-a suppressive factor in the macrophages mentioned above. In macrophages, LPS stimulation is mediated through a cell surface protein, CD14 (Wright et al., 1990). However, CD14 is a glycosylphosphatidylinositol-anchored protein without a transmembrane domain. Recent studies showed that one of the most important murine receptors of LPS is TLR4 (Chow et al., 1999; Hoshino et al., 1999; Takeuchi et al., 1999). TLR4 has been reported as a putative transmembrane receptor in mice, and it is responsible for the initiation of intracellular signal transduction after interaction with the LPS±CD14 or LTA±CD14 complex. In the experiment with TLR4 knock out mice (Takeuchi et al., 1999), it was revealed that not only LPS, but also LTA stimulation was mediated by TLR4. The C3H/HeJ mouse strain, characterized by hyporesponsiveness to LPS, has a single point mutation in the TLR 4 gene (Poltorak et al., 1998; Hoshino et al., 1999; Qureshi et al., 1999). The expression levels of both TNF-a and IL-1a mRNA in C3H/HeJ macrophages were lower than those in C3H/HeN macrophages in the presence of LPS (Fig. 6). Therefore, we hypothesize that LPS induced both TNF-a and IL-1 mRNA expression in the macrophages
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through TLR4. However, in LPS-stimulated macrophages from C3H/HeN mice, the TNF-a expression was suppressed with ES products, while the IL-1 gene expression was not changed. These data suggest that ES products inhibited TNF-a mRNA expression downstream from TLR4 in the LPS signal transduction. If CD14 or TLR4 were directly downregulated or blocked by ES products, IL-1 should also be suppressed. Although LPS-activated macrophages from C3H/HeJ mice express TNF-a and IL-1 mRNA, LTA-activated macrophages from C3H/HeJ did not express TNF-a or IL1 genes (Fig. 6). As for LPS receptors, macrophages have not only CD14±TLR4, but others (Hampton et al., 1991; Lynn and Golenbock, 1992; Ingalls and Golenbock, 1995; Ingalls et al., 1997; Medvedev et al., 1998). However, as for LTA-receptors, there has been no ef®cient receptor reported except TLR4. Therefore, TNF-a and IL-1 mRNA expression in LPS-activated macrophages from C3H/HeJ mice may be related to such receptors. Acknowledgements The authors are grateful to Professor Yuichi Ishibe, Department of Anesthesiology and Reanimatology, Faculty of Medicine, Tottori University, for his useful comments on the manuscript. This work was supported by a Grant-in-Aid for Scienti®c Research (numbers 09670258 and 11670241) from the Ministry of Science, Culture and Education, Japan. References Barrios-Rodiles, M., Tiraloche, G., Chadee, K., 1999. Lipopolysaccharide modulates cyclooxygenase-2 transcriptionally and posttranscriptionally in human macrophages independently from endogenous IL-1b and TNF-a. J. Immunol. 163, 963±9. Baumgartner, R.A., Deramo, V.A., Beaven, M.A., 1996. Constitutive and inducible mechanisms for synthesis and release of cytokines in immune cell lines. J. Immunol. 157, 4087±93. Berg, D.J., Kuhn, R., Rajewsky, K., Muller, W., Menon, S., Davidson, N., Grunig, G., Rennick, D., 1995. Interleukin-10 is a central regulator of the response to LPS in murine models of endotoxic shock and the Shwartzman reaction but not endotoxin tolerance. J. Clin. Invest. 96, 2339±47. Bogdan, C., Paik, J., Vodovotz, Y., Nathan, C., 1992. Contrasting mechanisms for suppression of macrophage cytokine release by transforming growth factor-b and interleukin-10. J. Biol. Chem. 267, 23301±8. Chantry, D., Turner, M., Abney, E., Feldmann, M., 1989. Modulation of cytokine production by transforming growth factor-b. J. Immunol. 142, 4295±300. Chaudhri, G., 1997. Differential regulation of biosynthesis of cell surface and secreted TNF-a in LPS-stimulated murine macrophages. J. Leukoc. Biol. 62, 249±57. Chow, J.C., Young, D.W., Golenbock, D.T., Christ, W.J., Gusovsky, F., 1999. Toll-like receptor-4 mediates lipopolysaccharide-induced signal transduction. J. Biol. Chem. 274, 10689±92. Clarke, C.J.P., Hales, A., Hunt, A., Foxwell, B.M.J., 1998. IL-10-mediated suppression of TNF-a production is independent of its ability to inhibit NF-kB activity. Eur. J. Immunol. 28, 1719±26. Descoteaux, A., Matlashewski, G., 1989. c-fos and tumor necrosis factor
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