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J. Biol. Chem., Vol. 277, Issue 39, 36825-36831, September 27, 2002

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Activation of Macrophages by Linear (13)--D-Glucans

IMPLICATIONS FOR THE RECOGNITION OF FUNGI BY INNATE IMMUNITY^*

Keiko Kataoka, Tatsushi Muta^§¶, Soh Yamazaki, and Koichiro Takeshige

From the  Department of Molecular and Cellular Biochemistry, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582 and ^§ Host and Defense, PRESTO, Japan Science and Technology Corporation (JST), Saitama 332-0012, Japan

Received for publication, July 8, 2002, and in revised form, July 24, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Although (13)--D-glucans, which are one of major fungal cell wall components, are known to activate invertebrate innate immune systems, their activities on mammalian cells remain elusive. Here, we report their activities on mouse macrophages. Among the various (13)--D-glucans, curdlan, a linear (13)--D-glucan, although not branched -glucans, exhibits significant activity to stimulate nuclear factor-B in macrophages. The activity of curdlan is dramatically enhanced by pretreatment with sodium hydroxide or dimethyl sulfoxide, which disrupts multiple-stranded helices of (13)--D-glucans, and is dose-dependently inhibited by a (13)--D-glucan-binding protein and by laminarioligosaccharides with (13)--D-glucosidic linkages. Intriguingly, the activity of curdlan is also augmented by incubation with zymolyase, which releases (13)--D-glucans with a single helical structure from the glucan-networks assembled by multiple-stranded helices. The activation of macrophages culminates in the production of inducible nitric-oxide synthase, tumor necrosis factor-, and macrophage inflammatory protein-2. Furthermore, a dominant-negative mutant of MyD88, an adaptor protein mediating signaling through the Toll-like receptor/inerleukin-1 receptor-like (TIR) domain, inhibits the activation of macrophages by curdlan. These results strongly suggest that macrophages respond to linear (13)--D-glucans, possibly released from fungal cell walls, via a receptor(s) harboring the TIR domain, such as a Toll-like receptor, to induce inflammatory reactions.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Innate immune systems respond to characteristic molecules on microorganisms. Such molecules are indispensable structural components for the survival of microorganisms, and their presence in large numbers makes them ideal targets for recognition by innate immune systems, which utilize limited numbers of germ line-encoded proteins. The target molecules, represented by lipopolysaccharide (LPS)¹ and peptidoglycan (PGN) on Gram-negative and -positive bacteria, are called pathogen-associated molecular patterns (PAMPs) (1, 2). Recent studies have revealed that diverse molecules on bacteria or virus, including lipoprotein/peptides, flagellin, CpG DNA, and double-stranded RNA, function as PAMPs to stimulate the mammalian innate immune system via specific Toll-like receptors (TLRs), mammalian homologues of the Drosophila membrane protein Toll (3-9). Despite the intensive investigation of the responses to bacteria, much less is known about the responses to fungi, another important pathogen for multicellular organisms. The phagocytic responses and subsequent inflammatory reactions of macrophages to zymosan, a yeast cell wall component, indicate that the mammalian innate immune system has the capacity to respond to fungi (10). However, identification of PAMPs on fungi remains elusive because zymosan is a crude mixture of glucans, mannan, proteins, chitin, and glycolipids (11).

Among the fungal cell wall components, glucans with (13)--glucosidic linkages, (13)--D-glucans, are most abundantly present and provide mechanical strength to the cell walls (12). In addition to fungi, (13)--D-glucans are widely distributed in algae and higher plants, but rarely found in animals (12). In invertebrates, (13)--D-glucans are known to be potent stimulators for the innate immune system. In horseshoe crabs, factor G, a (13)--D-glucan-sensitive serine protease, is activated by the glucan to induce hemolymph coagulation (13-17). (13)--D-Glucans also activate the prophenoloxidase-activating cascade that leads to melanin formation in insects and crayfish (18). In plants, (13)--D-glucans are one of the elicitors to induce phytoalexin production (19, 20).

In mammals, (13)--D-glucans are known to be potent activators of the complement system (21). Furthermore, previous reports indicate that (13)--D-glucans exert inhibitory activities against tumor growth (22-25), in addition to exhibiting anti-inflammatory activities (26, 27). Because these activities are considered to be expressed through the stimulation of the reticulo-endothelial system, including stimulation of macrophages, endothelial, and reticulum cells, (13)--D-glucans are known as biological response modifiers (BRMs). In contrast to these activities observed in vivo, however, relatively little is known about the activities of (13)--D-glucans in vitro, and hence the molecular mechanisms of the cellular activation by (13)--D-glucans are poorly understood. Although some reports have described that (13)--D-glucans elicit the production of cytokines and nitric oxide (28-30), the relationships between the structures of the glucans and their stimulatory activities are still controversial due to a lack of reproducible in vitro systems to evaluate the activities of (13)--D-glucans.

In the present study, we evaluate the activities of (13)--D-glucans with a nuclear factor-B (NF-B) reporter system constructed with macrophages. This reliable and reproducible in vitro system allows characterization of the responses of macrophages to (13)--D-glucans. The results obtained indicate that the linear (13)--D-glucan curdlan exhibits significant cell-stimulating activities, and that the activities of (13)--D-glucans are dependent on their lengths and conformations. Furthermore, the analysis of the intracellular signaling suggests the involvement of MyD88, and therefore of a Toll/interleukin-1 receptor-like (TIR) domain-containing receptor(s), in the responses to (13)--D-glucans.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Reagents-- Curdlan, LPS from Escherichia coli 0111:B4, PGN from Staphylococcus aureus, and laminarin from Laminaria digitata were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), List Biological Laboratories, Inc. (Campbell, CA), Fluka Chemika-Biochemika (Buchs, Switzerland), and Sigma Chemical Co., respectively. Polymyxin B sulfate, poly(I)·poly(C), and mouse interferon- were from Sigma, Amersham Biosciences, and Genzyme Corp. (Cambridge, MA), respectively, and laminarioligosaccharides and zymolyase were from Seikagaku Corp. (Tokyo, Japan). LPS concentration in curdlan was measured by means of an LPS-specific Limulus amebocyte lysate (LAL) test using an Endospecy kit (Seikagaku Corp.). Glutathione S-transferase (GST) and a GST fusion protein with the xylanase Z-like domain of the horseshoe crab factor G were prepared as described previously (15). An expression plasmid for a dominant-negative mutant for MyD88 (pcDNA3-hMyD88C) was constructed by inserting a cDNA fragment for human MyD88 (amino acid 155 to the COOH terminus) created by a polymerase chain reaction into pcDNA3 (Invitrogen Life Technologies, Carlsbad, CA). pELAM1-Luc (31) and pRL-TK (Promega Corp., Madison, WI) were used as an NF-B reporter plasmid and an internal control plasmid, respectively.

Cells-- RAW264.7 and RAW-R12 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% heat-inactivated fetal calf serum (FCS) supplemented with 100 units/ml penicillin and 100 µg/ml streptomycin. RAW-R12 cells were obtained by stably transfecting pELAM-1-Luc into RAW264.7 cells.

NF-B Reporter Assay-- RAW-R12 cells (1 × 10⁵ cells/well), stably transfected with the NF-B reporter plasmid pELAM1-Luc, were seeded in a 96-well plate on the day before stimulation. The cells were stimulated as indicated and lysed, and their luciferase activities were measured by using a luciferase assay system (Promega Corp.). RAW264.7 cells (5× 10⁵ cells/well) in a 24-well plate were transfected with an expression plasmid together with an NF-B reporter, pELAM1-Luc, and a control Renilla luciferase reporter, pRL-TK, using FuGENE 6^TM transfection reagent according to the manufacturer's instructions (Roche Diagnostics). The cells were stimulated as indicated and lysed, and their luciferase activities were measured by using a dual-luciferase reporter assay system (Promega Corp.). The NF-B reporter activity was divided by the activity of the Renilla control reporter to normalize transfection efficiency.

Western Blotting-- Cells were stimulated as indicated and lysed in a buffer containing 50 mM Tris-HCl (pH 7.5), 0.15 M NaCl, 1% Nonidet P-40, 2 mM EDTA, 50 mM NaF, and 0.2 mM Na₃VO₄, supplemented with Complete^TM protease inhibitor mixture (Roche Diagnostics) on ice for 30 min. After centrifugation, the cell lysate was immunoblotted with anti-inducible nitric-oxide synthase (iNOS) monoclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

Tumor Necrosis Factor (TNF)- Bioassay-- TNF- secreted from stimulated cells into culture medium was assayed by bioassay using L929 cells (32). The standard curve was obtained with recombinant mouse TNF- (Genzyme Techne). It was confirmed that more than 95% of TNF- activities in the sample were neutralized with anti-mouse TNF- monoclonal antibody (Genzyme Techne).

Northern Blot Analysis-- After RAW264.7 cells were stimulated, total RNA was extracted from the cells by using TRIzol^TM (Invitrogen Life Technologies). 10 µg of RNA was subjected to electrophoresis on a 1.2% agarose gel, transferred to Hybond^TM-N+ nylon membrane (Amersham Biosciences), and hybridized with a ³²P-labeled probe for iNOS, TNF-, or macrophage inflammatory protein-2 (MIP-2). As a loading control, the same blot was hybridized with a probe for glyceraldehyde-3-phosphate dehydrogenase.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Curdlan, a Linear (13)--D-Glucan Preparation, Activates Macrophages-- To evaluate the activity of (13)--D-glucans on the mammalian cells, we assessed NF-B activation in macrophages, which is one of the prominent responses of the mammalian innate immune system. Among various (13)--D-glucan preparations examined, curdlan, a linear (13)--D-glucan derived from Alcaligenes faecalis var. myxogenes 10C3K (33), at final concentrations of 10-100 µg/ml, induced robust activation of NF-B in the mouse macrophage cell line RAW264.7, which was stably transfected with an NF-B reporter plasmid (RAW-R12) (Fig. 1A). Because curdlan is insoluble at neutral pH, it was once solubilized in NaOH solution and then added to the culture medium to neutralize pH by diluting 100-fold. The NaOH-pretreated curdlan was added to the cells for stimulation immediately after the neutralization. When the contamination of the curdlan preparation with LPS, a strong activator of macrophages, was measured by the LPS-specific Limulus amebocyte lysate (LAL) test, it was revealed that <1 pg of LPS was present per microgram of curdlan (data not shown). To rule out the possibility that the NF-B-stimulating activity was due to contaminating LPS, the cells were stimulated in the presence of polymyxin B, which neutralizes the LPS activity (34). Polymyxin B effectively inhibited the activity of 100 ng/ml LPS (Fig. 1B), but not the activity of curdlan (Fig. 1A). In all of the following experiments, stimulation by curdlan was done in the presence of polymyxin B. 

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Curdlan stimulates NF-B activity of macrophages. RAW-R12 cells (1 × 105 cells), stably transfected with an NF-B reporter, were stimulated by curdlan (A) or LPS (B) at the indicated concentrations in the presence (+) or absence () of 100 units/ml polymyxin B (Pmx). Curdlan was dissolved in 0.1 M NaOH and diluted 100-fold with DMEM containing 10% FCS to give the indicated final concentration before addition to the cells. After incubation at 37 °C for 6 h, the cells were lysed and the luciferase activities were measured. Relative activities to that of unstimulated cells are shown. Data are shown as the mean ± S.E. of duplicate samples and are representative of two independent experiments.

We carefully titrated the concentrations of NaOH used for solubilizing curdlan (Fig. 2A). Curdlan was solubilized with NaOH at the indicated concentrations, then diluted 100-fold with the medium and used to stimulate the cells. NaOH alone at 0.01-0.3 M did not stimulate the NF-B activity (data not shown). Insoluble curdlan suspension in water (0 M NaOH) showed weak activity, and this activity was significantly augmented by increasing the concentrations of NaOH. The addition of 0.15 M NaOH to the medium raised the pH of the medium by 0.2-0.3 units, but the viability of the cells did not change during the assay (data not shown). Although the activity was reduced with 0.3 M NaOH, this reduction was most likely caused by the toxic effects of the increased pH of the medium on the cells: when NaOH-pretreated curdlan was diluted 1,000-fold with the medium, the NF-B-stimulating activity of curdlan pretreated with 0.3 M NaOH was higher than that of curdlan pretreated with 0.15 M NaOH (data not shown). When LPS was similarly treated with NaOH, its ability to activate the NF-B of macrophages was gradually reduced by increasing concentrations of NaOH (Fig. 2B). The NF-B-stimulating activity of PGN, which is insoluble even in the NaOH solutions, was scarcely affected by NaOH, but was also inhibited at higher NaOH concentrations (Fig. 2C). When KOH was used instead of NaOH, similar augmentation of the curdlan activity and inhibition of the LPS and PGN activities were observed (data not shown). We also found that another preparation of a linear (13)--D-glucan, paramylon, activated the cells to a lesser degree, and in an NaOH-dependent manner like that of curdlan (data not shown). Curdlan solubilized in dimethyl sulfoxide (Me₂SO) also exhibited the enhanced activity as in NaOH (Fig. 2D).

View larger version (28K): [in this window] [in a new window]   Fig. 2.   The activity of curdlan is enhanced by pretreatment with NaOH or Me2SO. Curdlan (A), LPS (B), or PGN (C) was dissolved in NaOH at the indicated concentrations. Then, the samples were diluted 100-fold with DMEM containing 10% FCS before stimulation of RAW-R12 cells (1 × 105 cells), stably transfected with an NF-B reporter. In D, curdlan or LPS was dissolved in 0.15 M NaOH or Me2SO and then diluted 100-fold with the medium. To simultaneously compare the effects of the pretreatment with NaOH and Me2SO, the stimulants pretreated with NaOH or Me2SO were diluted with the medium containing Me2SO or NaOH, respectively, to keep the final concentrations of NaOH and Me2SO constant. In the experiments without the pretreatment (None), the stimulants in water were diluted with the medium containing both NaOH and Me2SO. The final concentrations of NaOH and Me2SO in the medium were kept constant. The final concentrations of curdlan, LPS, and PGN were 100 µg/ml, 1 ng/ml, and 100 µg/ml, respectively. Stimulation by curdlan was carried out in the presence of 100 units/ml polymyxin B. After incubation at 37 °C for 6 h, the cells were lysed and the luciferase activities were measured. Relative activities to that of unstimulated cells are shown. Data are shown as the mean ± S.E. of duplicate samples and are representative of three independent experiments.

We next tried to activate the insoluble curdlan without the pretreatment with NaOH or Me₂SO, since these compounds do not occur under physiological conditions. We tested the effect of treatment with zymolyase, an endoglucosidase, which partially solubilizes curdlan. Zymolyase treatment for 15-30 min markedly stimulated the activity of curdlan (Fig. 3A). Longer treatment with zymolyase failed to completely solubilize curdlan, the activity of which was slightly reduced at 60 min and sustained up to 24 h. The effect of zymolyase treatment was specific to curdlan, because zymolyase itself did not activate NF-B in the absence of curdlan (data not shown), and the enzyme treatment did not affect the activity of LPS (Fig. 3B). When the zymolyase-treated curdlan was centrifuged, the activity was found in the insoluble fraction, and the soluble fraction showed little activity (Fig. 3C). Zymolyase treatment of paramylon for 15 min also enhanced its NF-B-stimulating activity, which was gradually decreased by longer treatment with zymolyase (data not shown).

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Zymolyase treatment of curdlan enhances its activity. Curdlan (A) or LPS (B) was dissolved in phosphate-buffered saline with or without 0.5 mg/ml zymolyase (Zym) and incubated at 37 °C for the indicated time. Then the zymolyase-treated curdlan and LPS were diluted 100-fold with DMEM containing 10% FCS to give the final concentrations of 100 µg/ml and 1 ng/ml, respectively, and added to RAW-R12 cells (1 × 105 cells), stably transfected with an NF-B reporter. C, curdlan treated with zymolyase for 30 min was centrifuged at 20,000 × g for 15 min to obtain the soluble fraction (Sup). The resulting precipitates (Ppt) were washed three times with phosphate-buffered saline and then suspended in the original volume of the same buffer. The obtained soluble and insoluble fractions were diluted 100-fold with the medium and used to stimulate the cells. Stimulation by curdlan was carried out in the presence of 100 units/ml polymyxin B. After incubation at 37 °C for 4 h, the cells were lysed, and the luciferase activities were measured. Relative activities to that of unstimulated cells are shown. Data are shown as the mean ± S.E. of duplicate samples and are representative of four independent experiments.

Because the NaOH and zymolyase treatments convert curdlan from insoluble particulates to soluble glucans, it is possible that these treatments release unknown contaminants in the particulates that activate macrophages. We attempted to determine if the macrophage-stimulating activity is due to (13)--D-glucan itself by using a (13)--D-glucan-binding protein. The COOH-terminal xylanase Z-like domain of the (13)--D-glucan-sensitive horseshoe crab factor G binds specifically to a (13)--D-glucan disaccharide at a K_a of 5.77 × 10⁷ M¹ and neutralizes the activity of (13)--D-glucan to activate factor G (15). We utilized this glucan-binding domain (GBD) of the horseshoe crab factor G to examine the specificity of the activity of curdlan. When preincubated with a glutathione S-transferase (GST) fusion protein containing GBD (GST-GBD), the activity of NaOH-pretreated curdlan was dose-dependently inhibited (Fig. 4A). The inhibition was not observed with GST. In contrast to the effect on curdlan, the activity of LPS was not affected either by GST-GBD or GST (Fig. 4B). Neither GST nor GST-GBD activated NF-B in the absence of curdlan (data not shown). Thus, it was confirmed that the activity of curdlan was indeed due to the authentic (13)--D-glucan.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   A -glucan-binding protein inhibits the activity of curdlan. Curdlan pretreated with 0.15 M NaOH (A) or LPS (B) was diluted 100-fold with DMEM containing 10% FCS and the indicated concentrations of GST or a GST fusion protein of GBD of horseshoe crab factor G (GST-GBD), and was incubated at 37 °C for 30 min. The final concentrations of curdlan and LPS were 100 µg/ml and 1 ng/ml, respectively. The preincubated curdlan or LPS was added to stimulate RAW-R12 cells (1 × 105 cells), stably transfected with an NF-B reporter, at 37 °C for 6 h. Then, the cells were lysed, and the luciferase activities were measured. Stimulation by curdlan was carried out in the presence of 100 units/ml polymyxin B. Relative activities to that of unstimulated cells are shown. Data are shown as the mean ± S.E. of duplicate samples and are representative of three independent experiments.

Curdlan-mediated Activation Is Inhibited by Shorter (13)--D-Glucans-- Because curdlan is a long (13)--D-glucan with a degree of polymerization (DP) of ~500, we next investigated the activities of short (13)--D-glucans. When the activities of laminarioligosaccharides, linear water-soluble (13)--D-glucans with a DP of 2-7, were examined by using RAW-R12 cells, none of them exhibited the stimulating activity at concentrations up to 1 mg/ml even after the NaOH pretreatment (data not shown). Instead, laminarioheptaose (DP = 7) inhibited the curdlan-mediated activation in a dose-dependent manner (Fig. 5A). Laminarioheptaose did not inhibit LPS-mediated activation, however (Figs. 5B and D). When laminarioligosaccharides with different lengths were examined, the inhibition was dependent on the DP of the oligosaccharides, with the longer oligosaccharides exhibiting the greater inhibition (Fig. 5C). We further examined the activity of laminarin derived from L. digitata, which is a water-soluble (13)--D-glucan preparation with a DP of 20-30, and with branches of single glucosyl residues by a (16)--linkage (35). Laminarin exhibited a weak stimulating activity at 1-5 mg/ml. Nevertheless, it dose-dependently inhibited the curdlan-mediated activation without affecting the LPS-mediated activation (Fig. 5, E and F).

View larger version (34K): [in this window] [in a new window]   Fig. 5.   Short (13)--D-glucans inhibit the activity of curdlan. RAW-R12 cells (1 × 105 cells), stably transfected with an NF-B reporter, were preincubated with DMEM containing 10% FCS and glucose or laminarioheptaose (Heptaose) (A and B), laminarioligosachharides with varying DP (1 mg/ml) (C and D), or laminarin (E and F) at 37 °C for 30 min. Then, curdlan pretreated with 0.15 M NaOH (A, C, and E) or LPS (B, D, and F) was directly added to the medium to give the final concentration of 100 µg/ml or 1 ng/ml, respectively. In the experiments in E and F, 0.15 M NaOH alone was used as a negative control (None). After incubation at 37 °C for 6 h, the cells were lysed and the luciferase activities were measured. Stimulation by curdlan was carried out in the presence of 100 units/ml polymyxin B. Relative activities to that of unstimulated cells are shown. Data are shown as the mean ± S.E. of duplicate samples and are representative of four independent experiments.

Curdlan Induces Production of Proinflammatory Mediators-- We next investigated whether the stimulation of macrophages with curdlan leads to inflammatory reactions. We treated thioglycolate-elicited peritoneal macrophages with curdlan or LPS and examined the induction of iNOS by Western blotting. 16 h after stimulation with curdlan, a significant induction of iNOS was observed (Fig. 6A). Like the induction by LPS, the induction by curdlan was considerably enhanced by co-stimulation with interferon-. Polymyxin B inhibited the induction by LPS but not that by curdlan. The induction of iNOS was also observed in bone marrow-derived macrophages (data not shown) and RAW264.7 cells (Fig. 6B) by the treatment with curdlan alone. When the time course of the iNOS induction was examined, the induction by curdlan was much slower than that by LPS (Fig. 6B). We also measured the TNF- production in the cells stimulated by curdlan or LPS. In contrast to the induction of iNOS, curdlan alone induced TNF- secretion comparable to that by LPS with a similar time course (Fig. 6C).

View larger version (30K): [in this window] [in a new window]   Fig. 6.   Curdlan induces iNOS and TNF-. A, thioglycolate-elicited peritoneal macrophages were stimulated with 100 µg/ml curdlan pretreated with 0.15 M NaOH or 1 ng/ml LPS in the presence (+) and absence () of 10 units/ml interferon- (IFN-) and 100 units/ml polymyxin B (Pmx) at 37 °C for 16 h. B, RAW264.7 cells were stimulated with 100 µg/ml curdlan pretreated with 0.15 M NaOH or 1 ng/ml LPS at 37 °C for the indicated time. Then the cell lysate was prepared and analyzed by Western blotting with an anti-iNOS antibody. C, RAW264.7 cells were stimulated with 100 µg/ml curdlan pretreated with 0.15 M NaOH or 100 ng/ml LPS for the indicated time. TNF- in the culture supernatant was measured by an L929 cytotoxicity assay. Data are shown as the mean ± S.E. of duplicate samples. D, curdlan, LPS, and PGN were pretreated with the indicated concentrations of NaOH. They were then diluted 100-fold with DMEM containing 10% FCS and added to RAW264.7 cells for stimulation at 37 °C for 16 h. The final concentrations of curdlan, LPS, and PGN were 100 µg/ml, 1 ng/ml, and 100 µg/ml, respectively. The cell lysate was analyzed by Western blotting with an anti-iNOS antibody. Data shown are representative of at least three independent experiments.

When the effect of the NaOH pretreatment on the iNOS induction was examined, the induction was found to be dependent on the concentrations of NaOH: the maximum induction was observed when curdlan was pretreated with 0.15 M NaOH (Fig. 6D). The activity of LPS was reduced by higher concentrations of NaOH and that of PGN was less affected. These results coincide well with the effect of NaOH on the NF-B activation.

To determine whether the inductions by curdlan occur at the mRNA level, we analyzed changes of mRNAs for iNOS and TNF- after the stimulation (Fig. 7). The induction of iNOS mRNA by curdlan was slower and much weaker compared with that by LPS. On the other hand, the curdlan-stimulated cells exhibited a rapid and strong induction of TNF- mRNA, which was comparable to that of the LPS-stimulated cells. These results are consistent with the pattern of the protein expression shown in Fig. 6. We also found that mRNA for macrophage inflammatory protein (MIP)-2, another inflammatory mediator, was also induced by curdlan as strongly as by LPS. In contrast to mRNA for iNOS or TNF-, the MIP-2 mRNA decayed faster in curdlan-stimulated cells than in LPS-stimulated cells.

View larger version (88K): [in this window] [in a new window]   Fig. 7.   Curdlan induces mRNAs for iNOS, TNF-, and MIP-2. RAW264.7 cells were stimulated with 100 µg/ml curdlan pretreated with 0.15 M NaOH or 1 ng/ml LPS for the indicated time. Total RNA was extracted from the cells and subjected to Northern blotting analysis with a probe for iNOS, TNF-, MIP-2, or glyceraldehydes-3-phosphate dehydrogenase (GAPDH). Data shown are representative of two independent experiments.

Cellular Activation by Curdlan Is Mediated through a MyD88-dependent Pathway-- Curdlan stimulation of macrophages culminates in the production of inflammatory mediators that are also produced by LPS- or PGN-stimulation, whose signaling is transduced into the cells through TLR4 or TLR2, respectively (4, 36, 37). Although TLR2- or TLR4-transfected HEK293 cells did not respond to curdlan,² we suspected that curdlan might activate other TLRs. All the TLRs thus far identified utilize the adaptor protein MyD88. The truncated mutant of MyD88 harboring the COOH-terminal region interacts with the cytoplasmic domain of TLRs and hence acts as a dominant-negative mutant (38, 39). We used this mutant to determine whether curdlan-mediated signaling is also mediated by MyD88. When the activation of NF-B was evaluated on vector- or the MyD88 mutant-transfected RAW264.7 cells, the curdlan-mediated activation was significantly inhibited in the mutant-transfected cells, as were the LPS- and PGN-mediated activations (Fig. 8). On the other hand, transfection of the MyD88 mutant was ineffective in inhibiting the activation by the double-stranded RNA poly(I)·poly(C), as shown in previous reports (40, 41). The results indicate that curdlan-mediated signaling is also mediated through the MyD88-dependent pathway.

View larger version (18K): [in this window] [in a new window]   Fig. 8.   Curdlan-mediated signaling is inhibited by a dominant-negative mutant of MyD88. RAW264.7 cells were transfected with an expression plasmid for a dominant-negative mutant of MyD88 (MyD88C) or an empty vector (Vector), together with an NF-B reporter plasmid, pELAM1-Luc, and a control Renilla reporter plasmid, pRL-TK. The cells were stimulated with 100 µg/ml curdlan pretreated with 0.15 M NaOH, 10 µg/ml poly(I)·poly(C) (pIpC), 1 ng/ml LPS, or 100 µg/ml PGN at 37 °C for 6 h. Then the cells were lysed, and the luciferase activities were measured. The transfection efficiency was normalized by Rennilla luciferase activity derived from co-transfected control vector, pRL-TK. Percent activities to that of vector-transfected cells are shown. Data are shown as the mean ± S.E. of duplicate samples and are representative of three independent experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

After screening of various (13)--D-glucans by measuring NF-B-activating activity in macrophages, we identified curdlan, a linear (13)--D-glucan, as the strongest activator. The glucans screened by the current system included branched (13)--D-glucans such as laminarin, paramylon, schizophyllan, lentinan, bakers' yeast -glucan, barley -glucan, and krestin. Although schizophyllan, lentinan, and krestin are known as BRM and have been reported to induce the production of nitric oxide and/or TNF- in macrophages (28, 42, 43), by our present assay system we could not detect significant NF-B-stimulating activities of these branched (13)--D-glucans at concentrations of 100 µg/ml with or without NaOH pretreatment. Instead, another linear (13)--D-glucan, paramylon, activated NF-B after the pretreatment with NaOH, but to a much lesser extent than curdlan.

Various microbe-derived molecules have been reported to activate innate immunity via respective specific TLRs (2). We carefully examined the possibility that this activity was due to a trace amount of contaminants derived from microbes in the curdlan preparation. Based on the following observations, we concluded that the activity of curdlan shown in the present study was due to the authentic (13)--D-glucan. The activity of curdlan was not affected by polymyxin B, which neutralized 100 ng/ml LPS effectively (Fig. 1). The activity of curdlan was dependent on the pretreatment with NaOH, whereas those of LPS and PGN were not affected or reduced by the pretreatment (Fig. 2). Furthermore, TLR2-, 3-, 4-, 5-, or 9-transfected HEK293 cells, which are responsive to PGN/lipopeptides, double-stranded RNA, LPS, flagellin, or CpG DNA, respectively (7-9, 31, 44, 45), did not respond to curdlan.² More convincing evidence was obtained when we tested the effect of the glucan-binding protein. GBD of the horseshoe crab factor G, which specifically binds to (13)--D-glucosidic linkages, inhibited the activity of curdlan but not that of LPS (Fig. 4). The modulatory effects of short (13)--D-glucans and zymolyase, an endo-(13)--D-glucosidase, on the activity of curdlan further supported the conclusion. Since this was the first careful examination of such contaminants, and since our results showed that some preparations of -glucans contained polymyxin B-inhibitable activities (data not shown), a portion of the activities of (13)--D-glucans or their derivatives described in previous reports might be attributable to the contaminant(s) that stimulates macrophages.

Zymosan, which consists of yeast cell wall particles, is one of the strong macrophage activators containing (13)--D-glucans. In contrast to curdlan, zymosan activated TLR2-transfected HEK293 cells, and the activation of macrophages by zymosan was only weakly inhibited by the short (13)--D-glucans or GBD (data not shown). Thus, zymosan should contain multiple stimulators for macrophages in addition to (13)--D-glucans, and hence it is not an ideal material to study the activities of (13)--D-glucans.

The activity of curdlan was dramatically enhanced by the pretreatment with NaOH (Figs. 2 and 6D). Long gel-forming (13)--D-glucans are basically composed of a single helix in water, and the single helices of these molecules associate to form local junction zones composed of double- or triple-stranded helices, which are connected by the single helices to form networks (46). The addition of NaOH up to 0.19 M gradually breaks the multiple-helical structures without changing the single helical conformation. At NaOH concentrations between 0.19 and 0.22 M, a transition occurs from the single helix to random structures (47, 48). Pretreatment with Me₂SO, which converts the conformation of curdlan to random structures (49), also activated curdlan (Fig. 2D). The augmentation of the activity of curdlan by pretreatment with NaOH or Me₂SO indicates that the single helix and random structures are active conformations for the stimulation of NF-B in macrophages. It has been reported that the horseshoe crab factor G is also activated by (13)--D-glucans with the single helical and random structures, but not by those with triple-stranded helices (50).

The zymolyase treatment, which is unlikely to change the conformation of curdlan, would release the single helices from the network of curdlan, since the enzyme is an endoglucanase that cleaves the single helices but not the multiple helices (51). The observation that the zymolyase treatment stimulated the activity of curdlan indicates that the molecular flexibility of the single helix, which is fixed by the multiple-stranded helices, is important for the activity of curdlan. Solubility seems not to be important for the activity of (13)--D-glucans, because even after the zymolyase treatment, the active fraction was in the insoluble fraction but not in the soluble fraction (Fig. 3D). Carboxymethylated curdlan is soluble in water, but inactive in our assay system (data not shown).

The activity of curdlan to stimulate NF-B was inhibited by transfection of a MyD88 dominant-negative mutant containing the COOH-terminal TIR domain (Fig. 8). MyD88 acts as an essential adaptor protein proximal to interleukin-1 receptor and TLRs by associating the receptors through their TIR domains. Therefore, the inhibition by the MyD88 mutant strongly suggests that the signal transduction induced by (13)--D-glucans is mediated through a member(s) of the TLR family. Although several membrane components, such as complement receptor 3 (52), a scavenger receptor (53), lactosylceramide (54), and dectin-1 (55, 56), have been reported to bind to (13)--D-glucans, none of these proteins are likely to be inhibited by the MyD88 mutant. They might function as phagocytic receptors, although their significance in the activation of macrophages is unknown. The identification of the responsible TIR-containing receptor(s) for curdlan remains to be determined. The qualitatively different induction patterns of iNOS, TNF-, and MIP-2 by curdlan (Figs. 6 and 7) suggest that the receptor for curdlan is distinct from that for LPS or PGN.

As in the cases of most PAMPs that stimulate TLRs, it is currently unknown whether or not curdlan directly binds to a cell surface receptor. Our characterization of curdlan-mediated activation of macrophages, however, provided some insight into the nature of the curdlan-recognizing protein. Water-soluble short linear (13)--D-glucan oligosaccharides with a DP of <7 did not activate macrophages at all; in fact, they inhibited the curdlan-mediated activation (Fig. 5). Thus, it is strongly suggested that the short (13)--D-glucans with the random structures bind to the curdlan-recognizing protein or receptor, but are too short to support the activation of this protein. Since linear (13)--D-glucans with a DP of <20 are soluble, and the soluble fraction of the zymolyase-treated curdlan did not exhibit the NF-B- activating activity; the active glucans should have DPs of more than 20. Branched (13)--D-glucans as long as curdlan were incapable of inducing the NF-B activation. It has been shown that activation of the horseshoe crab factor G is induced by the intermolecular interaction of factor G molecules on a template of linear and sufficiently long (13)--D-glucans that support the collision of the molecules (14, 15). The curdlan-recognizing protein or receptor might be activated by a mechanism similar to that of factor G, thereby leading to the activation of macrophages.

Although many reports have described the activity of -glucans as BRM in vivo, much less is known about the in vitro activity of (13)--D-glucans. This fact suggests that a complex system is required to express the activity of (13)--D-glucans. As we showed in this paper, the macrophage-stimulating activity of curdlan requires disassembly or disruption of the multiple-stranded helices at junction zones of the linear (13)--D-glucans. From a physiological or pathological point of views, it is an intriguing finding that curdlan was activated by zymolyase. On fungal cell surfaces, (13)--D-glucans exist as an insoluble matrix immobilized on the cell walls, which could be enzymatically released by a glucan-hydrolase(s) such as (13)--, (16)--, or (14)--D-glucosidase. The released fragments may be the activators for macrophages. In addition, some carrier proteins for the released insoluble fragments might be present. If the cells producing such glucosidases and/or carrier proteins are different from the cells activated by the modified (13)--D-glucans, it is not hard to imagine that a series of reactions would be difficult to reconstitute in the in vitro systems. Elucidation of the physiological or pathological modification of (13)--D-glucans, and the identification of the receptor responsible for the glucan-mediated signaling should lead to a better understanding of the responses of the innate immune system to fungi, as well as the development of a new therapeutic utilization of (13)--D-glucans as an effective BRM.

	ACKNOWLEDGEMENTS

We thank Y. Sunakawa for expert technical assistance and J. Aketagawa (Seikagaku Corp.) for helpful comments and discussion. We also thank H. Tamura (Seikagaku Corp.), Kaken Chemical Co., Ltd., Yamanouchi Pharmaceutical Co., Ltd., and Sankyo Co., Ltd., for providing the Endospecy kit, schizophyllan, lentinan, and krestin, respectively.

	FOOTNOTES

^* This study was supported by grants-in-aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (to T. M. and K. T.), and grants from the Sumitomo Foundation (to T. M.), the Naito Foundation (to T. M.), the Mochida Memorial Foundation for Medical and Pharmaceutical Research (to T. M.), and the Kaibara Foundation (to T. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed. Tel.: 81-92-642-6103; Fax: 81-92-642-6103; E-mail: tmuta@mailserver.med.kyushuu.ac.jp.

Published, JBC Papers in Press, July 24, 2002, DOI 10.1074/jbc.M206756200

² K. Kataoka and T. Muta, unpublished data.

	ABBREVIATIONS

The abbreviations used are: LPS, lipopolysaccharide; PGN, peptidoglycan; PAMP, pathogen-associated molecular pattern; TLR, Toll-like receptor; BRM, biological response modifier; NF-B, nuclear factor-B; TIR, Toll/interleukin-1 receptor-like; GST, glutathione S-transferase; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; iNOS, inducible nitric-oxide synthase; TNF, tumor necrosis factor; MIP-2, macrophage inflammatory protein-2; Me₂SO, dimethyl sulfoxide; GBD, glucan-binding domain; DP, degree of polymerization.

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