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Involvement of TGF-β in Inhibitory Effects of Negatively Charged Liposomes on Nitric Oxide Production by Macrophages Stimulated with LPS
Biochemical and Biophysical Research Communications 281, 614 – 620 (2001) doi:10.1006/bbrc.2001.4419, available online at http://www.idealibrary.com on
Involvement of TGF- in Inhibitory Effects of Negatively Charged Liposomes on Nitric Oxide Production by Macrophages Stimulated with LPS Ryozo Matsuno, Yukihiko Aramaki, 1 and Seishi Tsuchiya Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan
Received January 29, 2001
We examined the role of TGF- in the inhibitory effects of negatively charged liposomes composed of phosphatidylserine (PS-liposomes) on nitric oxide (NO) production by macrophages stimulated with LPS. The expression of TGF- mRNA increased when mouse peritoneal macrophages were treated with PSliposomes. The inhibitory effect of PS-liposomes on NO production was restored by treatment with antiTGF- antibody. Furthermore, NO production, iNOS mRNA expression, and iNOS protein induction by LPS were inhibited by treatment of macrophages with TGF- as well as PS-liposomes. These results indicated that PS-liposomes down-regulate NO production by macrophages through the induction of TGF- and suggested that TGF- may suppress NO production upstream of the transcription of iNOS mRNA. © 2001 Academic Press
Key Words: liposome; macrophage; nitric oxide; TGF; p38 MAP kinase.
Nitric oxide (NO) is a short-lived radical gas implicated in multiple biological processes including vascular homeostasis, neurotransmission, and antimicrobial defense (1). Three isoforms of nitric oxide synthase (NOS) have been identified in mammalian cells (2– 4). Type I NOS (nNOS) and Type III NOS (eNOS) are constitutively expressed in neural and endothelial cells, respectively, and synthesize NO in response to external stimuli. On the other hand, Type II NOS (iNOS) found in multiple cell types, including macrophages, hepatocytes, endothelial cells, and smooth muscle cells is induced in response to various stimuli such as interferon-␥ (IFN-␥), interleukin-1 (IL-1 ), tumor necrosis factor-␣ (TNF-␣) and bacterial lipopolysaccharide (LPS), and NO production is regulated at the transcriptional level. NO produced by iNOS has 1
To whom correspondence should be addressed. Fax: (⫹81)42676-3182. E-mail: [email protected]. 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
antimicrobial and tumoricidal effects (5– 8). However, the overproduction of NO can be detrimental to the host due to its nonspecific cytotoxicity. Therefore, precise regulation of NO production is critical for preservation of the normal function of the host defense system and the survival of host cells. We previously reported that negatively charged liposomes composed of phosphatidylserine (PS-liposomes) inhibited NO production from thioglycollate-elicited mouse peritoneal macrophages stimulated with LPS, and this was due to the inhibition of iNOS induction, but not inhibition of iNOS activity (9). Furthermore, pretreatment of macrophages with PS-liposomes was required for the inhibition of NO production (9). However, the mechanism by which PS-liposomes inhibit iNOS induction in macrophages remains unclear. Fadok et al. reported that phagocytosis of apoptotic neutrophils inhibited the production of cytokines such as interleukin (IL)-1, IL-8, IL-10, granulocytemacrophage colony-stimulating factor (GM-CSF) and TNF-␣ in human monocyte-derived macrophages, and indicated that secretion of transforming growth factor- (TGF-), prostaglandin E 2 (PGE 2), and/or platelet-activating factor (PAF) following the phagocytosis of apoptotic cells could lead to these inhibitory effects (10, 11). Since apoptotic cells expose PS on the outer leaflet of the plasma membrane, and are removed by macrophages through several kinds of receptors such as scavenger receptors (12–15), the secretion of these substances from macrophages would be expected to inhibit NO production following the phagocytosis of PS-liposomes. TGF- is a multifunctional polypeptide growth factor that has been increasingly recognized as an important immunoregulatory molecule, although its reported effects on immunological responses are often contradictory (16, 17). TGF- was shown previously to downregulate the production of proinflammatory mediators in macrophages (18 –20). Therefore, we examined the induction of TGF- following PS-liposome treatment,
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and then clarified whether TGF- contributes to the suppression of iNOS expression and NO production in macrophages stimulated with LPS. MATERIALS AND METHODS Materials. phosphatidylcholine (PC) from egg yolk was purchased from Nippon Oil and Fat Co., Ltd. (Tokyo, Japan). Lipopolysaccharide (LPS) from Escherichia coli (serotype 0111:B4), and PS from calf brain were purchased from Sigma Co., Ltd. (St. Louis, MO). Mouse anti-TGF- monoclonal antibody was purchased from Genzyme (Cambridge, MA). Human platelet TGF- was purchased from Habbor Bio-Products (Norwood, MA). Preparation of liposomes. Multilamellar liposomes were prepared by vortexing and passed through a membrane filter (0.45 m; Corning Glassworks, Corning, NY) before use. Lipid compositions of liposomes were PS:PC:cholesterol ⫽ 2:1:1 (PS-liposomes) and PC: cholesterol ⫽ 3:1 (PC-liposomes). LPS contamination in the liposome preparation and culture media was estimated using a Limulus amebocyte lysate assay as a routine laboratory practice (Wako Pure Chemicals Co., Ltd., Osaka, Japan), and was less than 18 pg/ml. Preparation of macrophages. C3H/HeN mice (male, 6 – 8 weeks old, Japan SLC Inc., Shizuoka, Japan) were injected intraperitoneally with 1.0 mL of 3% thioglycollate (Difco Laboratory, Detroit, MI). On day 4, the peritoneal macrophages were prepared according to our previous method (9). Preparation of macrophage culture supernatants. Macrophages (1 ⫻ 10 5 cells/well) were treated with liposomes (500 g as lipid/ml) for 24 h and culture supernatants were collected by ultracentrifugation (100,000g, 30 min). Nitrite determination. Macrophages (1 ⫻ 10 cells/well) were treated with liposomes (500 g as lipid/ml), culture supernatants, or TGF-. Macrophages were then further incubated for 48 h with LPS (10 g/ml) to elicit the production of NO. NO production was estimated by measurement of nitrite in the culture supernatant using Griess reagent as described by Stuehr and Nathan (21). Macrophage viability subsequent to this treatment was assessed by the Trypan blue dye exclusion test, and was shown to have not changed. 5
Western blotting analysis. Macrophages (1 ⫻ 10 6 cells/dish) were treated with liposomes (500 g as lipid/ml) or TGF- (10 ng/ml), and further incubated with LPS (10 g/ml) for specified times. Cells were then lysed by our previous method (9). For determination of iNOS protein, cell lysate (15 g as protein) was separated by 7.5% SDS– PAGE, blotted on Immobilon P membranes (Nihon Millipore, Tokyo, Japan) and analyzed using rabbit anti-iNOS antibody (Affinity BioReagent Inc., Neshanic Station, NJ) for 2 h. Membranes were incubated with peroxidase-conjugated goat anti-rabbit antibody (Cappel, Durham, NC), and specific bands were detected with an ECL assay kit (Amersham Japan, Tokyo, Japan). Band intensity was analyzed with NIH Image (22). For determination of p38 and phosphorylated p38, samples (15 g as protein) were separated by 12% SDS–PAGE, blotted on membranes and analyzed using anti-p38 monoclonal antibody (SC-535, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and a Phospho Plus p38 MAP kinase (Tht-180/Tyr-182) Antibody Kit (New England BioLabs, Beverly, MA), respectively. RT-PCR analysis. iNOS and TGF- gene expressions were detected by RT-PCR (23). Total RNA (2 g) was isolated from macrophages with Isogen solution (Nippon Gene, Toyama, Japan). cDNAs were synthesized by SuperScript II. Then, cDNAs were amplified with specific primers for iNOS and TGF-. -actin primers were used as an internal control. Primers were designed based on the mouse sequences (iNOS: forward, 5⬘-ACAGGGAAGTCTGAAGCACTAG-3⬘, reverse, 5⬘CATGCAAGGAAGGGAACTCTTC-3⬘; TGF-: forward, 5⬘-CTTTAGGAAGGACCTGGGTT-3⬘, reverse, 5⬘-CAGGAGCGCACAATCATGTT-3⬘;
FIG. 1. Effects of liposomes on LPS-induced production of NO from macrophages. Peritoneal macrophages (1 ⫻ 10 5 cells/well) were incubated with liposomes (500 g/ml) or culture supernatants of macrophages incubated with liposomes for 24 h and further incubated for 48 h with or without LPS (10 g/ml) to elicit NO. The supernatants of macrophages were collected and nitrite levels were measured as described in the Methods section. The values are the means ⫾ SD of triplicate cultures from three independent experiments.
-actin: forward, 5⬘-GCACCACACCTTCTACAATGAG-3⬘, reverse, 5⬘TTGGCATAGAGGTCTTTACGGA-3⬘). PCR was performed for 32 cycles of denaturation at 94°C for 60 s, annealing at 55°C for 120 s, and extension at 72°C for 180 s. The amplified products were analyzed on 1.5% agarose gels containing 0.1 g/ml ethidium bromide, and bands were visualized with UV. Band intensity was analyzed with NIH Image (22).
RESULTS AND DISCUSSION Production of the Inhibitory Factor by PS-Liposomes We previously reported (9) that PS-liposomes inhibited NO production from mouse peritoneal macrophages stimulated with LPS, and pretreatment of macrophages with PS-liposomes appeared to be required for the inhibitory effect on NO production induced by LPS; no effect was observed when PS-liposomes were added to macrophage cultures at the same time as LPS stimulation. We thus speculated that a factor that interferes with NO production may be secreted from macrophages following PS-liposome treatment. Macrophages were treated with liposomes for 24 h, and cul-
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FIG. 2. Involvement of TGF- in inhibitory effects of PS-liposomes on NO production. (A) Effect of PS-liposomes on the expression of TGF- mRNA. Macrophages (1 ⫻ 10 6 cells/dish) were treated with liposomes (500 g/ml) for the indicated times. Total RNAs were extracted, and then RT-PCR was performed. (B) Effect of anti-TGF- antibody on NO production. Macrophages (1 ⫻ 10 5 cells/well) were incubated with PS-liposomes (500 g/ml) for 24 h in the absence or presence of anti-TGF- antibody (50 g/ml). Macrophages were then further incubated with or without LPS (10 g/ml) for 48 h. The supernatants of macrophage cultures were collected and nitrite levels were measured.
ture supernatant was collected by ultracentrifugation, then the supernatant was added to the naive macrophages and incubated with LPS for 48 h. As shown in Fig. 1, NO production induced by LPS was only inhibited when the macrophages were treated with the culture supernatant prepared by incubation with PSliposomes. The intensity of inhibition was almost the same as that of the positive control macrophages that were treated with PS-liposomes directly. These results
suggested that macrophages secrete an inhibitory factor and this factor interferes with NO production from macrophages stimulated with LPS. Induction of TGF- by PS-Liposomes Apoptotic cells show cell-surface changes that allow recognition and removal by macrophages (24, 25). Removal occurs before lysis, which prevents the release of
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potentially toxic and immunogenic intracellular contents from the apoptotic cells into the surrounding tissue, and inflammation is avoided (26). For the removal of apoptotic cells, macrophages recognize PS on these cells and phagocytose them through CD36 as the predominant scavenger receptor (12, 27). As a consequence, macrophages produce TGF-, PGE 2 and PAF, and these products inhibit the production of inflammatory cytokines such as IL-1␣, IL-8, TNF-␣ and GM-CSF from macrophages stimulated with LPS (10, 11). TGF- is a multifunctional polypeptide growth factor that has been increasingly recognized as an important immunoregulatory molecule, although its reported effects on immunological responses are often contradictory (16, 17). TGF- was shown previously to downregulate the production of proinflammatory mediators in macrophages (18 –20). Therefore, we turned our attention to TGF- as a factor that may modulate NO production from macrophages following PS-liposome treatment, and its production was evaluated by RTPCR analysis. Figure 2A shows the expression of TGF- mRNA following treatment of macrophages with liposomes. TGF- mRNA was constitutively expressed in macrophages, its level began to increase 1 h after addition of PS-liposomes, reaching a maximum at 3 h, and gradually decreased to the control level thereafter. No changes in TGF- mRNA levels were observed in macrophages treated with PC-liposomes, however, which showed the same levels as controls throughout the experimental period. Furthermore, the effects of anti-TGF- antibody on NO production from macrophages were examined; NO production was restored by treatment with this antibody (Fig. 2B). These findings suggested that TGF- may be the factor that suppresses NO production from macrophages stimulated with LPS. Suppression of NO Production by TGF- Since PS-liposomes were shown to inhibit the expression of iNOS mRNA and iNOS protein and to result in the suppression of NO production from macrophages stimulated with LPS in our previous studies (9), we examined whether the addition of TGF- to LPS-stimulated macrophages caused the inhibition of NO production by the same mechanism as PSliposomes. NO production from macrophages was inhibited by TGF- in a saturable manner, but the inhibitory effect of TGF- was not complete even when macrophages were treated with 25 ng/ml of TGF- (Fig. 3). There have been several reports (18 –20) indicating that TGF- can inhibit NO production from macrophages. Vodovotz et al. (20) also reported the inhibitory activity of TGF- on NO production from macrophages, and suggested that the potency of TGF- reflects the suppression of NO production by three distinct mech-
FIG. 3. The effect of TGF- on NO production from macrophages stimulated with LPS. Macrophages (1 ⫻ 10 5 cells/well) were incubated with various concentrations of TGF- for 2 h, and further incubated with or without LPS (10 g/ml) for 48 h. The supernatants of macrophage cultures were collected and nitrite levels were measured. The values are means ⫾ SD (n ⫽ 3).
anisms; decreased stability and translation of iNOS mRNA and increased degradation of iNOS protein. Therefore, we first compared the effects of TGF- on iNOS mRNA expression in LPS-stimulated macrophages with those of liposomes using RT-PCR assay. LPS caused an increase in iNOS mRNA expression, which reached a steady- state amount at 6 h, and the expression of iNOS mRNA was inhibited by pretreatment of macrophages with TGF- as well as PSliposomes, but not PC-liposomes (Fig. 4A). Next the effects of TGF- on the induction of iNOS protein were measured by Western blotting analysis. Consistent with results regarding NO production and iNOS mRNA expression, iNOS protein induction by LPS was also inhibited by addition of TGF- as well as PSliposomes, but not PC-liposomes (Fig. 4B). These findings suggested that macrophages secrete TGF- following treatment with PS-liposomes, which resulted in the suppression of LPS-induced NO production. Effects of TGF- on Activation of p38 MAP Kinase Mitogen activated protein (MAP) kinases are comprised of three principal family members, i.e., extracellular-signal regulated kinase (ERK), c-Jun
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FIG. 5. The effects of TGF- on LPS-induced phosphorylation of p38 MAP kinase in mouse peritoneal macrophages. Macrophages (1 ⫻ 10 6 cells/dish) were treated with TGF- (10 ng/ml) for the indicated times, then stimulated with LPS (10 g/ml) for 15 min. Cell lysates were analyzed by Western blotting using anti-mouse p38 MAP kinase antibody (lower panel) and a Phospho Plus p38 MAP kinase Antibody Kit (upper panel).
NH 2-terminal kinase or stress activated protein kinase (JNK/SAPK), and p38 MAP kinase, each of which has different isoforms (28). Although LPS stimulated all MAP kinases in macrophages, it has only been reported that p38 MAP kinase contributes to LPSinduced NO production (28, 29). Recently, we investigated the effects of PS-liposomes on p38 MAP kinase to clarify the mechanisms of the inhibitory effects on NO production, and found that PS-liposomes inhibited LPS-induced activation of p38 MAP kinase in macrophages (30). Furthermore, NO production from macrophages stimulated by LPS was completely inhibited by SB203580, a specific inhibitor of p38 MAP kinase (30). These findings indicated that the inhibition of p38 MAP kinase activation was crucial in the suppression of NO production from macrophages treated with PSliposomes. To clarify whether the inhibition of p38 MAP kinase activation by PS-liposomes was cause by TGF-, the effects of TGF- on p38 MAP kinase activation were examined. As p38 MAP kinase is activated by dual phosphorylation of tyrosine and threonine residues, the effects of TGF- on tyrosine phosphorylation of p38 MAP kinase were examined by Western blotting analysis using a Phospho Plus p38 MAP kinase (Tht180/Tyr-182) Antibody Kit. Tyrosine phosphorylation of p38 MAP kinase was observed after 15 min of stimulation with LPS, but unexpectedly TGF- did not inhibit LPS-induced phosphorylation of p38 MAP kinase (Fig. 5). These results indicated that TGF- is one of factors that inhibits NO production, and the contribution of another factor that interferes with NO production from macrophages treated with PS-liposomes was suggested.
On TGF- signaling, two distinct pathways, TAB1 (TAK binding protein 1)/TAK1 (TGF- activated kinase 1) pathway and Smad pathway, were well characterized (31, 32). TGF- has been reported to activate p38 MAP kinase though TAB1/TAK1 pathway in macrophages (31), but TGF- did not upregulate LPSinduced activation of p38 MAP kinase (Fig. 5). Therefore, the activation of p38 MAP kinase through TAB1/ TAK1 pathway is not involved in the suppression of NO production by TGF-. Most studies to date examining the function of Smad protein in TGF- signaling have focused on genes that are transactivated by TGF- (33–35). In contrast to TGF--mediated gene transactivation, the mechanism by which TGF- inhibits gene transcription is not well characterized. Recently, Werner et al. reported that Smat3 was a critical effector responsible for the inhibition of macrophage gene activation including iNOS by TGF-1, and suggested that competition for essential coactivators such as p300 might be an important determinant in regulating the degree of macrophage activation and inhibition by TGF- (36). Consequently, Smad pathway may act as important role in the suppression of iNOS expression by TGF- secreted from macrophages following PS-liposome treatment. In conclusion, the expression of TGF- mRNA was observed when mouse peritoneal macrophages were incubated with PS-liposomes, and NO production, iNOS mRNA expression and iNOS induction were inhibited by TGF-. These findings suggested that TGF- is the factor produced by PS-liposomes that suppresses NO production from macrophages stimulated with LPS. Studies are now underway to clarify
FIG. 4. The effects of liposomes and TGF- on LPS-induced (A) iNOS mRNA and (B) iNOS protein expression in mouse peritoneal macrophages. (A) Macrophages (1 ⫻ 10 6 cells/dish) were treated with PC- or PS-liposomes (500 g/ml) for 24 h or with TGF- (10 ng/ml) for 2 h and further incubated with LPS for the indicated times. Total RNAs were extracted, then RT-PCR was performed. (B) Macrophages (1 ⫻ 10 6 cells/dish) were treated with PC- or PS-liposomes (500 g/ml) for 24 h or with TGF- (10 ng/ml) for 2 h, and further incubated with LPS for the indicated times. Cell lysates were subjected to Western blotting as described under Materials and Methods. Band intensity was analyzed with NIH Image. 619
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how PS-liposomes affect macrophages to induce TGF- and how TGF- inhibits NO production. ACKNOWLEDGMENTS We are grateful to Mr. A. Uchida and Miss A. Kamiya for their technical assistance.
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17. Vodovotz, Y., Bogdan, C., Paik, J., Xie, Q. W., and Nathan, C. (1993) J. Exp. Med. 178, 605– 612. 18. Bogdan, C., Paik, J., Vodovotz, Y., and Nathan, C. (1992) J. Biol. Chem. 267, 23301–23308. 19. Imai, K., Takeshita, A., and Hanazawa, S. (2000) Infect. Immun. 68, 2418 –2423. 20. Vodovotz, Y., Letterio, J. J., Geiser, A. G., Chesler, L., Roberts, A. B., and Sparrow, J. (1996) J. Leukoc. Biol. 60, 261–270. 21. Stuehr, D. J., and Nathan, C. F. (1989) J. Exp. Med. 169, 1543– 1555. 22. Rasband, R. (1996) NIH Image (1.61) Manual 70 –76. 23. Arima, H., Takahashi, M., Aramaki, Y., Sakamoto, T., and Tsuchiya, S. (1998) Antisense Nucleic Acid Drug Dev. 8, 319 – 327. 24. Platt, N., da Silva, R. P., and Gordon, S. (1998) Trends in Cell Biol. 14, 365–372. 25. Fadok, V. A., Bratton, D. L., Rose, D. M., Pearson, A., Ezekewitz, R. A. B., and Henson, P. M. (2000) Nature 405, 85–90. 26. Savill, J. S., Wyllie, A. H., Henson, J. E., Walport, M. J., Henson, P. M., and Haslett, C. (1989) J. Clin. Invest. 83, 865– 875. 27. Fadok, V. A., Warner, M. L., Bratton, D. L., and Henson, P. M. (1998) J. Immunol. 161, 6250 – 6257. 28. Feng, G. J., Goodridge, H. S., Harnett, M. M., Wei, X. Q., Nikolaev, A. V., Higson, A. P., and Liew, F. Y. (1999) J. Immunol. 163, 6403– 6412. 29. Chen, C.-C., and Wang, J.-K. (1999) Mol. Pharmacol. 55, 481– 488. 30. Aramaki, Y., Matsuno, R., and Tsuchiya, S. Biochem. Biophys. Res. Commun., in press. 31. Shibuya, H., Yamaguchi, K., Shirakabe, K., Tonegawa, A., Gotoh, Y., Ueno, N., Irie, K., Nishida, E., and Matsumoto, K. (1996) Science 272, 1179 –1182. 32. Miyazono, K., ten Duke, P., and Heldin, C. H. (2000) Adv. Immunol. 75, 115–157. 33. Dennler, S., Itoh, S., Vivien, D., ten Dijke, P., Huet, S., and Gauthier, J. M. (1998) EMBO J. 17, 3091–3100. 34. Kon, A., Vindevoghel, L., Kouba, D. J., Fujimura, Y., Uitto, J., and Mauviel, A. (1999) Oncogene 18, 1837–1844. 35. Biggs, J. R., and Kraft, A. S. (1999) J. Biol. Chem. 274, 36987– 36994. 36. Werner, F., Jain, M. K., Feinberg, M. W., Sibinga, N. E., Pellacani, A., Wiesel, P., Chin, M. T., Topper, J. N., Perrella, M. A., and Lee, M. E. (2000) J. Biol. Chem. 275, 36653–36658.
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Involvement of TGF- in Inhibitory Effects of Negatively Charged Liposomes on Nitric Oxide Production by Macrophages Stimulated with LPS Ryozo Matsuno, Yukihiko Aramaki, 1 and Seishi Tsuchiya Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan
Received January 29, 2001
We examined the role of TGF- in the inhibitory effects of negatively charged liposomes composed of phosphatidylserine (PS-liposomes) on nitric oxide (NO) production by macrophages stimulated with LPS. The expression of TGF- mRNA increased when mouse peritoneal macrophages were treated with PSliposomes. The inhibitory effect of PS-liposomes on NO production was restored by treatment with antiTGF- antibody. Furthermore, NO production, iNOS mRNA expression, and iNOS protein induction by LPS were inhibited by treatment of macrophages with TGF- as well as PS-liposomes. These results indicated that PS-liposomes down-regulate NO production by macrophages through the induction of TGF- and suggested that TGF- may suppress NO production upstream of the transcription of iNOS mRNA. © 2001 Academic Press
Key Words: liposome; macrophage; nitric oxide; TGF; p38 MAP kinase.
Nitric oxide (NO) is a short-lived radical gas implicated in multiple biological processes including vascular homeostasis, neurotransmission, and antimicrobial defense (1). Three isoforms of nitric oxide synthase (NOS) have been identified in mammalian cells (2– 4). Type I NOS (nNOS) and Type III NOS (eNOS) are constitutively expressed in neural and endothelial cells, respectively, and synthesize NO in response to external stimuli. On the other hand, Type II NOS (iNOS) found in multiple cell types, including macrophages, hepatocytes, endothelial cells, and smooth muscle cells is induced in response to various stimuli such as interferon-␥ (IFN-␥), interleukin-1 (IL-1 ), tumor necrosis factor-␣ (TNF-␣) and bacterial lipopolysaccharide (LPS), and NO production is regulated at the transcriptional level. NO produced by iNOS has 1
To whom correspondence should be addressed. Fax: (⫹81)42676-3182. E-mail: [email protected]. 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
antimicrobial and tumoricidal effects (5– 8). However, the overproduction of NO can be detrimental to the host due to its nonspecific cytotoxicity. Therefore, precise regulation of NO production is critical for preservation of the normal function of the host defense system and the survival of host cells. We previously reported that negatively charged liposomes composed of phosphatidylserine (PS-liposomes) inhibited NO production from thioglycollate-elicited mouse peritoneal macrophages stimulated with LPS, and this was due to the inhibition of iNOS induction, but not inhibition of iNOS activity (9). Furthermore, pretreatment of macrophages with PS-liposomes was required for the inhibition of NO production (9). However, the mechanism by which PS-liposomes inhibit iNOS induction in macrophages remains unclear. Fadok et al. reported that phagocytosis of apoptotic neutrophils inhibited the production of cytokines such as interleukin (IL)-1, IL-8, IL-10, granulocytemacrophage colony-stimulating factor (GM-CSF) and TNF-␣ in human monocyte-derived macrophages, and indicated that secretion of transforming growth factor- (TGF-), prostaglandin E 2 (PGE 2), and/or platelet-activating factor (PAF) following the phagocytosis of apoptotic cells could lead to these inhibitory effects (10, 11). Since apoptotic cells expose PS on the outer leaflet of the plasma membrane, and are removed by macrophages through several kinds of receptors such as scavenger receptors (12–15), the secretion of these substances from macrophages would be expected to inhibit NO production following the phagocytosis of PS-liposomes. TGF- is a multifunctional polypeptide growth factor that has been increasingly recognized as an important immunoregulatory molecule, although its reported effects on immunological responses are often contradictory (16, 17). TGF- was shown previously to downregulate the production of proinflammatory mediators in macrophages (18 –20). Therefore, we examined the induction of TGF- following PS-liposome treatment,
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and then clarified whether TGF- contributes to the suppression of iNOS expression and NO production in macrophages stimulated with LPS. MATERIALS AND METHODS Materials. phosphatidylcholine (PC) from egg yolk was purchased from Nippon Oil and Fat Co., Ltd. (Tokyo, Japan). Lipopolysaccharide (LPS) from Escherichia coli (serotype 0111:B4), and PS from calf brain were purchased from Sigma Co., Ltd. (St. Louis, MO). Mouse anti-TGF- monoclonal antibody was purchased from Genzyme (Cambridge, MA). Human platelet TGF- was purchased from Habbor Bio-Products (Norwood, MA). Preparation of liposomes. Multilamellar liposomes were prepared by vortexing and passed through a membrane filter (0.45 m; Corning Glassworks, Corning, NY) before use. Lipid compositions of liposomes were PS:PC:cholesterol ⫽ 2:1:1 (PS-liposomes) and PC: cholesterol ⫽ 3:1 (PC-liposomes). LPS contamination in the liposome preparation and culture media was estimated using a Limulus amebocyte lysate assay as a routine laboratory practice (Wako Pure Chemicals Co., Ltd., Osaka, Japan), and was less than 18 pg/ml. Preparation of macrophages. C3H/HeN mice (male, 6 – 8 weeks old, Japan SLC Inc., Shizuoka, Japan) were injected intraperitoneally with 1.0 mL of 3% thioglycollate (Difco Laboratory, Detroit, MI). On day 4, the peritoneal macrophages were prepared according to our previous method (9). Preparation of macrophage culture supernatants. Macrophages (1 ⫻ 10 5 cells/well) were treated with liposomes (500 g as lipid/ml) for 24 h and culture supernatants were collected by ultracentrifugation (100,000g, 30 min). Nitrite determination. Macrophages (1 ⫻ 10 cells/well) were treated with liposomes (500 g as lipid/ml), culture supernatants, or TGF-. Macrophages were then further incubated for 48 h with LPS (10 g/ml) to elicit the production of NO. NO production was estimated by measurement of nitrite in the culture supernatant using Griess reagent as described by Stuehr and Nathan (21). Macrophage viability subsequent to this treatment was assessed by the Trypan blue dye exclusion test, and was shown to have not changed. 5
Western blotting analysis. Macrophages (1 ⫻ 10 6 cells/dish) were treated with liposomes (500 g as lipid/ml) or TGF- (10 ng/ml), and further incubated with LPS (10 g/ml) for specified times. Cells were then lysed by our previous method (9). For determination of iNOS protein, cell lysate (15 g as protein) was separated by 7.5% SDS– PAGE, blotted on Immobilon P membranes (Nihon Millipore, Tokyo, Japan) and analyzed using rabbit anti-iNOS antibody (Affinity BioReagent Inc., Neshanic Station, NJ) for 2 h. Membranes were incubated with peroxidase-conjugated goat anti-rabbit antibody (Cappel, Durham, NC), and specific bands were detected with an ECL assay kit (Amersham Japan, Tokyo, Japan). Band intensity was analyzed with NIH Image (22). For determination of p38 and phosphorylated p38, samples (15 g as protein) were separated by 12% SDS–PAGE, blotted on membranes and analyzed using anti-p38 monoclonal antibody (SC-535, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and a Phospho Plus p38 MAP kinase (Tht-180/Tyr-182) Antibody Kit (New England BioLabs, Beverly, MA), respectively. RT-PCR analysis. iNOS and TGF- gene expressions were detected by RT-PCR (23). Total RNA (2 g) was isolated from macrophages with Isogen solution (Nippon Gene, Toyama, Japan). cDNAs were synthesized by SuperScript II. Then, cDNAs were amplified with specific primers for iNOS and TGF-. -actin primers were used as an internal control. Primers were designed based on the mouse sequences (iNOS: forward, 5⬘-ACAGGGAAGTCTGAAGCACTAG-3⬘, reverse, 5⬘CATGCAAGGAAGGGAACTCTTC-3⬘; TGF-: forward, 5⬘-CTTTAGGAAGGACCTGGGTT-3⬘, reverse, 5⬘-CAGGAGCGCACAATCATGTT-3⬘;
FIG. 1. Effects of liposomes on LPS-induced production of NO from macrophages. Peritoneal macrophages (1 ⫻ 10 5 cells/well) were incubated with liposomes (500 g/ml) or culture supernatants of macrophages incubated with liposomes for 24 h and further incubated for 48 h with or without LPS (10 g/ml) to elicit NO. The supernatants of macrophages were collected and nitrite levels were measured as described in the Methods section. The values are the means ⫾ SD of triplicate cultures from three independent experiments.
-actin: forward, 5⬘-GCACCACACCTTCTACAATGAG-3⬘, reverse, 5⬘TTGGCATAGAGGTCTTTACGGA-3⬘). PCR was performed for 32 cycles of denaturation at 94°C for 60 s, annealing at 55°C for 120 s, and extension at 72°C for 180 s. The amplified products were analyzed on 1.5% agarose gels containing 0.1 g/ml ethidium bromide, and bands were visualized with UV. Band intensity was analyzed with NIH Image (22).
RESULTS AND DISCUSSION Production of the Inhibitory Factor by PS-Liposomes We previously reported (9) that PS-liposomes inhibited NO production from mouse peritoneal macrophages stimulated with LPS, and pretreatment of macrophages with PS-liposomes appeared to be required for the inhibitory effect on NO production induced by LPS; no effect was observed when PS-liposomes were added to macrophage cultures at the same time as LPS stimulation. We thus speculated that a factor that interferes with NO production may be secreted from macrophages following PS-liposome treatment. Macrophages were treated with liposomes for 24 h, and cul-
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FIG. 2. Involvement of TGF- in inhibitory effects of PS-liposomes on NO production. (A) Effect of PS-liposomes on the expression of TGF- mRNA. Macrophages (1 ⫻ 10 6 cells/dish) were treated with liposomes (500 g/ml) for the indicated times. Total RNAs were extracted, and then RT-PCR was performed. (B) Effect of anti-TGF- antibody on NO production. Macrophages (1 ⫻ 10 5 cells/well) were incubated with PS-liposomes (500 g/ml) for 24 h in the absence or presence of anti-TGF- antibody (50 g/ml). Macrophages were then further incubated with or without LPS (10 g/ml) for 48 h. The supernatants of macrophage cultures were collected and nitrite levels were measured.
ture supernatant was collected by ultracentrifugation, then the supernatant was added to the naive macrophages and incubated with LPS for 48 h. As shown in Fig. 1, NO production induced by LPS was only inhibited when the macrophages were treated with the culture supernatant prepared by incubation with PSliposomes. The intensity of inhibition was almost the same as that of the positive control macrophages that were treated with PS-liposomes directly. These results
suggested that macrophages secrete an inhibitory factor and this factor interferes with NO production from macrophages stimulated with LPS. Induction of TGF- by PS-Liposomes Apoptotic cells show cell-surface changes that allow recognition and removal by macrophages (24, 25). Removal occurs before lysis, which prevents the release of
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potentially toxic and immunogenic intracellular contents from the apoptotic cells into the surrounding tissue, and inflammation is avoided (26). For the removal of apoptotic cells, macrophages recognize PS on these cells and phagocytose them through CD36 as the predominant scavenger receptor (12, 27). As a consequence, macrophages produce TGF-, PGE 2 and PAF, and these products inhibit the production of inflammatory cytokines such as IL-1␣, IL-8, TNF-␣ and GM-CSF from macrophages stimulated with LPS (10, 11). TGF- is a multifunctional polypeptide growth factor that has been increasingly recognized as an important immunoregulatory molecule, although its reported effects on immunological responses are often contradictory (16, 17). TGF- was shown previously to downregulate the production of proinflammatory mediators in macrophages (18 –20). Therefore, we turned our attention to TGF- as a factor that may modulate NO production from macrophages following PS-liposome treatment, and its production was evaluated by RTPCR analysis. Figure 2A shows the expression of TGF- mRNA following treatment of macrophages with liposomes. TGF- mRNA was constitutively expressed in macrophages, its level began to increase 1 h after addition of PS-liposomes, reaching a maximum at 3 h, and gradually decreased to the control level thereafter. No changes in TGF- mRNA levels were observed in macrophages treated with PC-liposomes, however, which showed the same levels as controls throughout the experimental period. Furthermore, the effects of anti-TGF- antibody on NO production from macrophages were examined; NO production was restored by treatment with this antibody (Fig. 2B). These findings suggested that TGF- may be the factor that suppresses NO production from macrophages stimulated with LPS. Suppression of NO Production by TGF- Since PS-liposomes were shown to inhibit the expression of iNOS mRNA and iNOS protein and to result in the suppression of NO production from macrophages stimulated with LPS in our previous studies (9), we examined whether the addition of TGF- to LPS-stimulated macrophages caused the inhibition of NO production by the same mechanism as PSliposomes. NO production from macrophages was inhibited by TGF- in a saturable manner, but the inhibitory effect of TGF- was not complete even when macrophages were treated with 25 ng/ml of TGF- (Fig. 3). There have been several reports (18 –20) indicating that TGF- can inhibit NO production from macrophages. Vodovotz et al. (20) also reported the inhibitory activity of TGF- on NO production from macrophages, and suggested that the potency of TGF- reflects the suppression of NO production by three distinct mech-
FIG. 3. The effect of TGF- on NO production from macrophages stimulated with LPS. Macrophages (1 ⫻ 10 5 cells/well) were incubated with various concentrations of TGF- for 2 h, and further incubated with or without LPS (10 g/ml) for 48 h. The supernatants of macrophage cultures were collected and nitrite levels were measured. The values are means ⫾ SD (n ⫽ 3).
anisms; decreased stability and translation of iNOS mRNA and increased degradation of iNOS protein. Therefore, we first compared the effects of TGF- on iNOS mRNA expression in LPS-stimulated macrophages with those of liposomes using RT-PCR assay. LPS caused an increase in iNOS mRNA expression, which reached a steady- state amount at 6 h, and the expression of iNOS mRNA was inhibited by pretreatment of macrophages with TGF- as well as PSliposomes, but not PC-liposomes (Fig. 4A). Next the effects of TGF- on the induction of iNOS protein were measured by Western blotting analysis. Consistent with results regarding NO production and iNOS mRNA expression, iNOS protein induction by LPS was also inhibited by addition of TGF- as well as PSliposomes, but not PC-liposomes (Fig. 4B). These findings suggested that macrophages secrete TGF- following treatment with PS-liposomes, which resulted in the suppression of LPS-induced NO production. Effects of TGF- on Activation of p38 MAP Kinase Mitogen activated protein (MAP) kinases are comprised of three principal family members, i.e., extracellular-signal regulated kinase (ERK), c-Jun
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FIG. 5. The effects of TGF- on LPS-induced phosphorylation of p38 MAP kinase in mouse peritoneal macrophages. Macrophages (1 ⫻ 10 6 cells/dish) were treated with TGF- (10 ng/ml) for the indicated times, then stimulated with LPS (10 g/ml) for 15 min. Cell lysates were analyzed by Western blotting using anti-mouse p38 MAP kinase antibody (lower panel) and a Phospho Plus p38 MAP kinase Antibody Kit (upper panel).
NH 2-terminal kinase or stress activated protein kinase (JNK/SAPK), and p38 MAP kinase, each of which has different isoforms (28). Although LPS stimulated all MAP kinases in macrophages, it has only been reported that p38 MAP kinase contributes to LPSinduced NO production (28, 29). Recently, we investigated the effects of PS-liposomes on p38 MAP kinase to clarify the mechanisms of the inhibitory effects on NO production, and found that PS-liposomes inhibited LPS-induced activation of p38 MAP kinase in macrophages (30). Furthermore, NO production from macrophages stimulated by LPS was completely inhibited by SB203580, a specific inhibitor of p38 MAP kinase (30). These findings indicated that the inhibition of p38 MAP kinase activation was crucial in the suppression of NO production from macrophages treated with PSliposomes. To clarify whether the inhibition of p38 MAP kinase activation by PS-liposomes was cause by TGF-, the effects of TGF- on p38 MAP kinase activation were examined. As p38 MAP kinase is activated by dual phosphorylation of tyrosine and threonine residues, the effects of TGF- on tyrosine phosphorylation of p38 MAP kinase were examined by Western blotting analysis using a Phospho Plus p38 MAP kinase (Tht180/Tyr-182) Antibody Kit. Tyrosine phosphorylation of p38 MAP kinase was observed after 15 min of stimulation with LPS, but unexpectedly TGF- did not inhibit LPS-induced phosphorylation of p38 MAP kinase (Fig. 5). These results indicated that TGF- is one of factors that inhibits NO production, and the contribution of another factor that interferes with NO production from macrophages treated with PS-liposomes was suggested.
On TGF- signaling, two distinct pathways, TAB1 (TAK binding protein 1)/TAK1 (TGF- activated kinase 1) pathway and Smad pathway, were well characterized (31, 32). TGF- has been reported to activate p38 MAP kinase though TAB1/TAK1 pathway in macrophages (31), but TGF- did not upregulate LPSinduced activation of p38 MAP kinase (Fig. 5). Therefore, the activation of p38 MAP kinase through TAB1/ TAK1 pathway is not involved in the suppression of NO production by TGF-. Most studies to date examining the function of Smad protein in TGF- signaling have focused on genes that are transactivated by TGF- (33–35). In contrast to TGF--mediated gene transactivation, the mechanism by which TGF- inhibits gene transcription is not well characterized. Recently, Werner et al. reported that Smat3 was a critical effector responsible for the inhibition of macrophage gene activation including iNOS by TGF-1, and suggested that competition for essential coactivators such as p300 might be an important determinant in regulating the degree of macrophage activation and inhibition by TGF- (36). Consequently, Smad pathway may act as important role in the suppression of iNOS expression by TGF- secreted from macrophages following PS-liposome treatment. In conclusion, the expression of TGF- mRNA was observed when mouse peritoneal macrophages were incubated with PS-liposomes, and NO production, iNOS mRNA expression and iNOS induction were inhibited by TGF-. These findings suggested that TGF- is the factor produced by PS-liposomes that suppresses NO production from macrophages stimulated with LPS. Studies are now underway to clarify
FIG. 4. The effects of liposomes and TGF- on LPS-induced (A) iNOS mRNA and (B) iNOS protein expression in mouse peritoneal macrophages. (A) Macrophages (1 ⫻ 10 6 cells/dish) were treated with PC- or PS-liposomes (500 g/ml) for 24 h or with TGF- (10 ng/ml) for 2 h and further incubated with LPS for the indicated times. Total RNAs were extracted, then RT-PCR was performed. (B) Macrophages (1 ⫻ 10 6 cells/dish) were treated with PC- or PS-liposomes (500 g/ml) for 24 h or with TGF- (10 ng/ml) for 2 h, and further incubated with LPS for the indicated times. Cell lysates were subjected to Western blotting as described under Materials and Methods. Band intensity was analyzed with NIH Image. 619
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how PS-liposomes affect macrophages to induce TGF- and how TGF- inhibits NO production. ACKNOWLEDGMENTS We are grateful to Mr. A. Uchida and Miss A. Kamiya for their technical assistance.
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