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Biomacromolecules 2010, 11, 1212–1216
Branched Chitins as Nonnatural Immunomodulatory Biopolymers: Secretions of TNF-r and NO by Direct Stimulation of Macrophages Manabu Shimojoh,*,† Taku Kojima,‡ Kohji Nakajima,† Kiyoshige Hatta,† Akira Katoh,‡ and Keisuke Kurita*,‡ Research and Development Department, Toyo Suisan Kaisha, Ltd., Kohnan, Minato-ku, Tokyo 108-8501, Japan, and Department of Materials and Life Science, Faculty of Science and Technology, Seikei University, Musashino-shi, Tokyo 180-8633, Japan Received December 4, 2009; Revised Manuscript Received March 30, 2010
In view of the interesting properties of branched polysaccharides occurring in nature, biological activities of nonnatural branched chitins having β-1,6-N-acetyl-D-glucosamine branches on the poly(β-1,4-N-acetyl-Dglucosamine) backbone have been studied. The immunostimulatory activities of the branched chitins were determined and compared with those of lentinan, a β-1,3-D-glucan having β-1,6-D-glucose branches, using the mouse macrophagelike cell line RAW264.7 in vitro. The secretions of the tumor necrosis factor and nitric oxide proved to be significantly higher with the branched chitins than with lentinan. Moreover, when interferon-γ was used in conjunction with the branched chitins on macrophage treatment, a marked augmentation of nitric oxide production was observed. These results are interpreted as the direct stimulation of macrophages by the branched chitins, and the distinctive activities suggest the possibility of developing new types of polysaccharide antitumor agents.
Introduction Besides linear polysaccharides, various branched polysaccharides occur in nature. Some of these are known to exhibit significant biological activities. The properties of polysaccharides are sensitively dependent on the structures, and the presence of sugar branches is critical for expressing certain biological and physiological activities including antitumor activity.1,2 Typical examples are lentinan3 and schizophyllan,4 which are mushroom polysaccharides having the β-1,3-linked D-glucan main chain, that is, curdlan, with β-1,6-D-glucose branches, their branch numbers/main chain units being 2/5 and 1/3, respectively. These branched polysaccharides are particularly important because of their immunomodulating potential, which is not observed with curdlan. Therefore, they are currently used clinically as antitumor agents,5,6 though their water solubility is low. Despite the growing interest in antitumor agents, only limited attention has been paid to branched polysaccharides, probably because of the difficulty in isolating active polysaccharides and elucidating their structures. With regard to developing antitumor agents with high activity and yet allowably low toxicity, branched polysaccharides would be excellent candidates, but unfortunately, the number of promising polysaccharides and the amounts available for basic and clinical studies are quite low. A clue to overcome this problem may exist in a synthetic approach to obtain branched polysaccharides. It is particularly crucial to establish synthetic routes to well-defined molecular structures, which will enable an in-depth study of the relationship between activity and structure. * To whom all correspondence should be addressed. E-mail: shimojohm@ maruchan.co.jp (M.S.);
[email protected] (K.K.). † Toyo Suisan Kaisha. ‡ Seikei University.
Although chitin is a linear polysaccharide of N-acetyl-Dglucosamine connected in a β-1,4 manner, it exhibits a variety of interesting biological and physicochemical properties ascribable to the presence of acetylamino groups and would be useful in various fields such as medicine, toiletries, biotechnology, food, and agriculture.7-12 However, it remains an almost unutilized biomass resource due to the lack of solubility and poor chemical reactivity. In the course of our studies on this abundant and easily accessible biopolymer, our attention has been focused on the regioselective chemical modifications using 2-N-phthaloyl-chitosan as a key intermediate.13 Thus, sitespecific substitution of various groups has become possible as a result of successful discrimination of three kinds of functional groups,14 and the introduction of β-1,6-N-acetyl-D-glucosamine branches gave rise to the formation of nonnatural branched chitins with varying degrees of substitution.15 The synthesized branched chitins were highly soluble in water at neutral pH, unlike the original chitin that is insoluble in common solvents, and showed improved properties besides solubility with regard to moisture absorption, lysozyme susceptibility, and bactericidal activity. They may therefore be useful as new types of watersoluble chitins in various fields, in particular, for medicinal and cosmetic applications. Because of the presence of sugar branches and the architecture of chitin-based amino polysaccharides, the resulting branched chitins are expected to exhibit considerable antitumor activity, and moreover, they can be provided in quantity for clinical use as well as for basic studies. This prompted us to discuss in detail the influence of the chemical structure of some synthetic branched polysaccharides on the antitumor activity for the purpose of developing potent antitumor agents. In this paper we report the results of our studies on the activation behavior of branched chitins toward the mouse macrophagelike cell line RAW264.7 as evaluated from the secretions of tumor necrosis factor (TNF-R) and nitric oxide (NO).
10.1021/bm9013793 2010 American Chemical Society Published on Web 04/23/2010
Branched Chitins as Immunomodulatory Biopolymers
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Figure 1. Structures of branched chitin and lentinan.
Experimental Section Synthesis of Branched Chitins Having N-Acetyl-D-glucosamine Branches and Preparation of Branched Chitin Solutions. The introduction of N-acetyl-D-glucosamine branches into chitin was carried out by a series of modification reactions of chitosan involving Nphthaloylation, tritylation at C-6, acetylation at C-3, detritylation, glycosylation with an oxazoline derived from D-glucosamine at C-6, deprotection, and N-acetylation, according to a previously reported method.15 The degree of substitution (ds) of the final product was estimated from the C/N ratio of the elemental analysis of the glycosylated product, as mentioned in a previous paper.15 The molecular weight characteristics were determined by gel permeation chromatography (GPC) calibrated with pullulan standards purchased from Showa Denko K.K. Branched chitin (3 mg) was dissolved in a lactic acid solution prepared from 3 µL of 89% aqueous L-lactic acid and 3 mL of ultrapure water. After sonication for 1 h to ensure complete homogeneity, the pH of the solution was adjusted to 6.0 with 1 mol/L sodium hydroxide. The solution was sterilized by filtration with a 0.22 µm pore membrane filter. Cell Culture and Reagents. The mouse macrophagelike cell line RAW264.7 was obtained from the American Type Culture Collection, and the cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% (v/v) heat-inactivated fetal bovine serum (FBS). L929 cells were obtained from Riken Cell Bank and cultured in minimum Eagle’s medium (MEM) containing 2 mM L-glutamine and 10% heat-inactivated FBS. B16, Vero, Mv.1.Lu, and WI-38 cells were also obtained from Riken Cell Bank. B16, Vero, and WI-38 cells were cultured in MEM containing 2 mM L-glutamine and 10% heatinactivated FBS. Mv.1.Lu cells were cultured in MEM containing 2 mM L-glutamine, 1% (v/v) nonessential amino acids (NEAA), and 10% heat-inactivated FBS. DMEM, MEM, L-glutamine, and phosphatebuffered saline (-) (PBS(-)) were purchased from Nissui Pharmaceutical Co., Ltd., and FBS and NEAA were purchased from Life Technologies Japan, Ltd. Lentinan was purchased from Ajimonoto Co., Inc. The vial contained 1 mg of lentinan along with 100 mg of D-mannose and 2 mg of dextran 40. Under aseptic conditions, 2 mL of ultrapure water was added to the vial in this study. Recombinant murine TNF-R was purchased from PeproTECH EC, Ltd.; lipopolysaccharide (LPS from Escherichia coli 0111:B4) was obtained from Sigma-Aldrich Japan, Ltd.; and recombinant mouse interferon-γ (IFN-γ) was purchased from Cosmobio Co., Ltd. Actinomycin D, 25% (w/w) aqueous glutaraldehyde solution, sulfanilamide, naphthylethylene diamine dihydrochloride, phosphoric acid, and crystal violet were purchased from Wako Pure Chemical Industries, Ltd. All the cell assays were carried out at pH 7.1-7.4. Limulus Amebocyte Lysate Assays. The endotoxic activity of branched chitin solutions (1 mg/mL) was determined by a quantitative kinetic assay based on the reactivity of gram-negative endotoxin with Limulus amebocyte lysate (LAL) at 37 °C using test kits of Limulus ES-J Test Wako (Wako Pure Chemical Industries, Ltd.).16 The standard endotoxin was from E. coli (UKT-B, final concentration 0.0625-0.0078 EU/mL). Determination of TNF-r Secretion from RAW264.7 Cells. RAW264.7 cells were seeded in each well of 24-well plates (5 × 105/well) with 1 mL of DMEM containing 10% heat-inactivated FBS. After incubating the cells for 24 h at 37 °C, the branched chitin solution (final concentration 50 µg/mL) or lentinan solution (final concentration
50 µg/mL) was added to several wells, and the cells were incubated at 37 °C for 8 h. TNF-R secretion was measured using the L929 cell bioassay by a previously reported method17 as follows: L929 cells were seeded in each well of 96-well plates (1.5 × 104/well) with 50 µL MEM containing 10% heat-inactivated FBS. After 24 h at 37 °C, 25 µL of actinomycin D (4 µg/mL) was added to each well. A sample solution, which was obtained by dilution with MEM up to 625 times of the culture supernatant of RAW264.7 cells with the branched chitins or lentinan, or diluted TNF-R solution (0.0032-10 ng/mL in MEM) was then added (25 µL/well), and the cells were incubated at 37 °C in an atmosphere containing 5% CO2. After 24 h, the medium was removed, and the cells were washed with PBS(-). They were fixed with 100 µL/well of glutaraldehyde solution, which was obtained by diluting aqueous glutaraldehyde with PBS(-) 100 times and washed with water. A 0.2% crystal violet solution (w/v) in 2% ethanol solution (50 µL/ well) was added, and after 30 min, the cells were washed with water. The stained cells were lysed with 100 µL/well of 50% (v/v) ethanol containing 50 mM sodium dihydrogen phosphate, and the absorbance at 595 nm was measured (Bio-Kinetics Reader EL312e, BIO-TEK Instruments, Inc.). Assay for NO Secretion in RAW264.7 Cells. RAW264.7 cells were seeded in each well of 24-well plates (5 × 105/well) with 1 mL of DMEM containing 10% heat-inactivated FBS. After a 24 h incubation at 37 °C, a branched chitin solution (final concentration 50 µg/mL) or lentinan solution (final concentration 50 µg/mL) was added to several wells, and the cells were incubated for 24 h at 37 °C. NO secretion was assayed as follows: briefly, 100 µL of the supernatant was treated with an equal volume of the Griess reagent (2% sulfanilamide, 0.2% naphthylethylene diamine dihydrochloride, and 5% phosphoric acid)18 for 10 min at room temperature in the dark. The optical density of the mixture was measured at 540 nm, with sodium nitrite solutions (0.5-100 µM) being used as standards. Effect of IFN-γ on NO Secretion. RAW264.7 cells (5 × 105) were treated with LPS (2 ng/mL), IFN-γ (20 units/mL), or both. The cells (5 × 105) were also treated with a branched chitin (ds 0.22, 50 µg/ mL), IFN-γ (20 units/mL), or both. Similarly, they were treated with lentinan (50 µg/mL), IFN-γ (20 units/mL), or both. After 24 h at 37 °C, NO in the supernatant was determined using the Griess reagent. MTT Assay for Cytotoxic Effect. Cell-mediated reduction of 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Dojin Co., Ltd.) was assayed according to the method of Mosmann.19 A 50 µL cell suspension (3.0 × 103) was cultured in each well of 96-well plates and allowed to adhere overnight at 37 °C in an atmosphere containing 5% CO2. An equal volume of the branched chitin of ds 0.22 (500 µg/mL) or lentinan (500 µg/mL) was added to each well, and the cells were incubated for 24 h at 37 °C in an atmosphere containing 5% CO2. A stock MTT solution (5 mg/mL in PBS(-), 10 µL) was added, and the incubation was continued for an additional 4 h. The mixture was aspirated, and the cells were treated with 100 µL of dimethyl sulfoxide. The reduction of MTT was determined by measuring the absorbance at 540 nm. The percentage inhibition was calculated relative to the growth of the control cells handled similarly but without polysaccharide exposure.
Results and Discussion Branched Chitins. The structures of branched chitin and lentinan are indicated in Figure 1. The branched chitins were synthesized through a seven-step regioselective chemical modifica-
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Scheme 1. Synthetic Route to Branched Chitin
Table 1. Characteristics of Branched Chitins Used in this Study
c
dsa
Mnb × 10-3
Mwc × 10-3
Mw/Mnd
0.13 0.22 0.45 0.63
30.1 44.1 33.3 37.0
121 273 115 124
4.02 6.18 3.44 3.36
a Degree of substitution. b Number average molecular weight by GPC. Weight average molecular weight by GPC. d Polydispersity.
tion starting from fully deacetylated chitosan (Scheme 1), and the extent of branching was controlled by the reagent ratio in the glycosylation reaction.15 As summarized in Table 1, four kinds of branched chitins with different ds values were used in this study. Endotoxin Contained in Branched Chitin Solutions. Endotoxins directly stimulate macrophages to induce secretion of NO and cytokines including TNF-R, and thus endotoxin concentrations in the branched chitin solutions should be determined first. The detection limit of the LAL kit was less than 0.03 EU/mL as confirmed with the standard LPS. After incubation of a mixture of the LAL reagent and a branched chitin solution at 37 °C for 60 min, no aggregation was observed. This confirmed that the endotoxin concentrations of all branched chitin solutions were less than 0.03 EU/mL, and the influence by endotoxins could be excluded. Effect of Branched Chitins on TNF-r Secretion. The in vitro stimulation of macrophages obtained from a variety of tissues to develop cytotoxicity to tumor cells or microorganisms has been reviewed recently.20 The molecular actions during the triggering and expression of macrophage-mediated cytotoxicity are receiving considerable attention,21 and TNF-R was reported to be secreted by macrophages during the activation process.22,23 Macrophagelike cells RAW264.7 secrete TNF-R on certain stimulation, and thus RAW264.7 cells were subjected to treatment with the branched chitins. The amount of TNF-R could be determined with L929 cells that die out depending on the concentration of TNF-R. As represented in Figure 2, all the branched chitins significantly increased TNF-R production in RAW264.7 cells, which suggests the direct stimulation of RAW264.7 cells by the branched chitins. The amount of TNF-R secretion was influenced by the ds value, and the branched chitins of ds 0.22 and 0.45 were superior to the others in TNF-R secretion. Although polydispersity (Mw/ Mn) of the branched chitin of ds 0.22 was somewhat large compared to those of the other branched chitins, the extent of
branching seems to be an important factor for the activation judging from the comparably high levels of TNF-R secretion by branched chitins of ds 0.22 and 0.45. Naturally, the original chitin that is water-insoluble did not induced TNF-R secretion at all, supporting the importance of sugar side branches in expressing certain biological activities.2 The activity of lentinan was also examined for comparison, and only very little TNF-R secretion (ca. 0.02 ng/mL) was detected in the culture medium (Figure 2). The branched chitin of ds 0.22 was significantly more effective in TNF-R secretion than lentinan by approximately 250-fold at the same concentration. These results indicate that N-acetyl-D-glucosamine is much superior to D-glucose as a side chain unit for the activation of macrophages. Effect of Branched Chitins on NO Secretion. Activated macrophages produce NO and reactive oxygen species that play important roles in the host defense against tumor cells and microorganisms.24 The branched chitins were then evaluated in terms of the NO secretion by the stimulation of RAW264.7 cells. All the branched chitins increased the NO production by direct stimulation (Figure 3) in a manner similar to the secretion of TNF-R (Figure 2). The maximum NO secretion was achieved again at ds 0.22. In sharp contrast, lentinan induced TNF-R secretion at a low level (
