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Biological

Inflammatory Immune Responses by Water-insoluble {alpha}-glucans

S. Okamoto2,3, Y. Terao1, H. Kaminishi3, S. Hamada4 and S. Kawabata1,*

1 Department of Oral and Molecular Microbiology, Osaka University Graduate School of Dentistry, 1-8 Yamadaoka, Suita-Osaka 565-0871, Japan;
2 Laboratory of Virology and Vaccinology, National Institute of Biomedical Innovation, 7-6-8 Saito-Asagi, Ibaraki-Osaka 567-0085, Japan;
3 Department of Functional Bioscience, Fukuoka Dental College, 2-15-1 Tamura, Sawara-ku, Fukuoka 814-0193, Japan; and
4 Department of Life Science, Nihon University Advanced Research Institute for the Sciences and Humanities, Nihon University Kaikan Daini Bekkan, 12-5, Goban-cho, Chiyoda-ku, Tokyo 102-8251, Japan

Correspondence: * corresponding author, kawabata{at}dent.osaka-u.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Water-insoluble {alpha}-glucans are synthesized from sucrose by glucosyltransferase-I of mutans streptococci and play an important role in the development of dental plaque. Several types of β-glucans in fungal cell wall components and water-soluble {alpha}-glucans from Streptococcus mutans are known to modulate innate immunity. In the present study, we investigated whether water-insoluble {alpha}-glucans also induced inflammatory innate immune responses. Our results showed that water-insoluble {alpha}-glucans synthesized by Streptococcus sobrinus activated mouse peritoneal exudate macrophages to produce pro-inflammatory cytokines. The immunological responses were not due to contamination by sucrose, water-soluble {alpha}-glucan, lipopolysaccharide, or peptidoglycan. Furthermore, human monocytes stimulated by water-insoluble {alpha}-glucans produced TNF-{alpha} and IL-8, while human polymorphonuclear cells were activated by water-insoluble {alpha}-glucans, resulting in chemotaxis and hydrogen peroxide production. The results demonstrated that water-soluble {alpha}-glucans modulate macrophage- and granulocyte-induced inflammatory immune responses, and suggest that inflammation induced by those {alpha}-glucans is associated with the development of periodontal diseases.

Key Words: Streptococcus sobrinus • water-insoluble {alpha}-glucans • inflammatory immune responses


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mutans streptococci, including Streptococcus sobrinus, have been implicated as primary etiologic agents of dental caries in humans (Hamada and Slade, 1980; Loesche, 1986). These bacteria produce water-soluble and water-insoluble {alpha}-glucans from sucrose by multiple glucosyltransferases (GTFs), and S. sobrinus produces GTF-I, GTF-S1, GTF-S2, and GTF-S3. GTF-I is responsible for the generation of water-insoluble {alpha}-glucans, whereas GTF-S1, GTF-S2, and GTF-S3 synthesize water-soluble {alpha}-glucans (Abo et al., 1991; Hanada et al., 1993, 2002). These adhesive glucans, primarily water-insoluble {alpha}-glucans, contribute to the development of dental plaque, a type of biofilm composed of micro-organisms and their products. Oral bacteria adhere to tooth surfaces in the presence of dental plaque and release acids, which are important factors in their cariogenic potential (Hamada and Slade, 1980; Tamesada et al., 2004).

Clinical studies have shown that bacterial deposits in dental plaque are necessary to initiate the processes leading to periodontal diseases, as well as dental caries (Schuster, 1983). In addition, such deposits induce vascular changes typical of acute inflammatory reactions, resulting in vascular leakage of fluid, and the active migration of polymorphonuclear cells out of the vessels and into gingival tissues. Lymphocytes then accumulate adjacent to gingival squamous epithelium, and fibroblasts in the area begin to show morphologic changes. However, it remains unknown whether water-insoluble {alpha}-glucans also initiate these disease processes.

Recent studies have revealed that several types of β-glucans in fungal cell wall components modulate innate immunity by activating mouse macrophages via complement receptor 3, a scavenger receptor, and/or Toll-like receptors (TLRs) (Kataoka et al., 2002; Brown et al., 2003; Steele et al., 2003). These β-glucans also initiate inflammatory responses in macrophages via NF-{kappa}B activation, thus inducing fungi-mediated inflammatory and autoimmune diseases (Hahn et al., 2003; Lebron et al., 2003; Yoshitomi et al., 2005). In contrast, little is known regarding the biological activities of {alpha}-glucans. In a previous study, the administration of water-soluble {alpha}-glucans from S. mutans to mice activated peritoneal exudate macrophages, although the possibility of those functioning as pathogenic promoters was not discussed (Choi et al., 2005). Water-insoluble {alpha}-glucans are produced in the oral cavity, and adhere to gingival squamous tissues. If water-insoluble {alpha}-glucans, as well as β-glucans and water-soluble {alpha}-glucans, were shown to activate macrophages and induce inflammatory immune responses, water-insoluble {alpha}-glucans would be associated with the induction of inflammation of oral squamous tissues. In the present study, we examined whether water-insoluble {alpha}-glucans, as well as fungal cell wall β-glucans and water-soluble {alpha}-glucan, promote macrophage activation, causing inflammatory immune responses in mice and humans.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice, Cell Lines, and Reagents
Female BALB/c mice (from 6 to 12 wks old) were obtained from Charles River Japan (Yokohama, Japan). All aspects of the handling, care, and usage of animals were approved by the animal care committees of Osaka University and Fukuoka Dental College. A mouse macrophage cell line, J774.1, was cultured in complete RPMI 1640 medium as described previously (Okamoto et al., 2001). Dextran T-2000 was purchased from Amersham Biosciences Inc. (Piscataway, NJ, USA). LPS and β-1,3-glucan from Euglena gracilis were obtained from Sigma (St. Louis, MO, USA), and sucrose and peptidoglycan (type III) were purchased from Wako (Osaka, Japan). Muramyl dipeptide was purchased from the Peptide Institute (Osaka, Japan).

Bacteria, GTFs, Water-insoluble {alpha}-glucans, and Water-soluble {alpha}-glucans
S. sobrinus strain 6715 (Ooshima et al., 1993) was cultured in brain-heart infusion broth. Water-insoluble {alpha}-glucans were produced according to methods described previously (Koga et al., 1986; Hamada et al., 1989; Kawabata et al., 1993; APPENDIX 1). Water-soluble {alpha}-glucans were synthesized by S. sobrinus strain 6715 as described previously (Abo et al., 1991; Hanada et al., 1993, 2002). S. sobrinus cell wall extracts were purified as described previously (Hamada et al., 1971). The amount of LPS was determined by a Limulus test (Wako), and the amount of peptidoglycan or β-glucan was determined with a silkworm larva plasma test (Wako).

Preparation of Mouse Peritoneal Exudate Macrophages, Human Monocytes, and Polymorphonuclear Cells
We prepared mouse peritoneal exudate macrophages as described previously (Takeuchi et al., 1999). Human peripheral blood mononuclear cells were isolated as described previously (Huber-Lang et al., 2003), with informed consent obtained from each of the blood donors according to a protocol approved by the Osaka University Committee of Clinical Investigations. Human monocytes from peripheral blood mononuclear cells were purified with the use of a MACS Monocyte Isolation Kit (Miltenvi Biotec, Gladbach, Germany), and the purity of the cells was assessed by flow cytometry (FACScan, Becton-Dickinson, Franklin Lakes, NJ, USA). The purity of peritoneal exudate macrophages, polymorphonuclear cells, and human monocytes was 90%, 94%, and 92%, respectively.

Cell Proliferation
Splenocytes (4 x 105 cells/200 µL of complete RPMI 1640 medium) were stimulated for 4 days with the test materials in 96-well flat-bottomed microtiter plates. We assayed proliferative responses by treating the cells for the final 18 hrs of culture with [3H] thymidine (0.5 µCi/well; ICN, Irvine, CA, USA). Incorporated radioactivity was counted by means of a liquid scintillation counter.

Cytokine Measurement
Cytokine amounts in culture supernatants were determined with the use of enzyme-linked immunosorbent assay (ELISA) kits (Biosource International, Camarillo, CA, USA).

RNA Interference
Pre-designed small interfering (si)RNAs for TLR2, TLR4, and nucleotide-binding oligomerization domains (NOD)1 and NOD2 were purchased from Qiagen. TransIT-TKO transfection reagent (Mirus, Madison, WI, USA) was diluted with CD293 medium (Invitrogen, Carlsbad, CA, USA). siRNA was then mixed with the diluted transfection reagent and added to J774.1 cells. After incubation for 24 hrs, the expression levels of the targeted mRNAs were examined by reverse transcription (RT)-PCR (Uehara et al., 2005).

Chemotaxis Assay and Measurement of Hydrogen Peroxide Production
A chemotaxis assay and measurement of hydrogen peroxide production were performed as described previously (Terao et al., 2006; APPENDIX 2).

Statistical Evaluations
Data are presented as the means ± standard error (SE). Statistical analysis was performed by a non-parametric Mann-Whitney U test with Statview for Macintosh. All conclusions are based on a significance level of p < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Stimulation of Mouse Peritoneal Exudate Macrophages by Water-insoluble {alpha}-glucans
We examined the ability of different concentrations of water-insoluble {alpha}-glucans to promote the proliferation of mouse spleen cells. No proliferation of splenocytes was observed when the cells were treated for 4 days with concentrations ranging from 10 to 1000 µg/mL water-insoluble {alpha}-glucans in complete RPMI 1640 medium. In contrast, LPS dose-dependently stimulated the proliferation of these cells (Fig. 1AGo). Next, we examined the effects of water-insoluble {alpha}-glucans on the production of pro-inflammatory cytokines by mouse peritoneal exudate macrophages. Cytokine ELISA results revealed that peritoneal exudate macrophages produced TNF-{alpha} and IL-6, but not IL-1β, following stimulation with water-insoluble {alpha}-glucans, whereas no such production was seen with sucrose or water-soluble {alpha}-glucans from S. sobrinus (Figs. 1B, 1C, 1DGo). The cytokine production of peritoneal exudate macrophages stimulated with water-insoluble {alpha}-glucan was significantly greater than that by stimulation with muramyl dipeptide, or with cell wall extracts from S. sobrinus, which contained lipoteichoic acid and peptidoglycan (APPENDIX 3). Since water-insoluble {alpha}-glucans are soluble in NaOH, we also determined the effect of solubilization with NaOH. Water-insoluble {alpha}-glucans (3, 10, 30, or 100 mg) were solubilized with 1 N of NaOH, diluted 1000-fold with medium, and then added to peritoneal exudate macrophages. Cytokine production by the peritoneal exudate macrophages was not significantly changed by suspension of water-insoluble {alpha}-glucans in PBS or 1 N of NaOH (APPENDIX 4). In addition, it was revealed that NaOH alone did not induce cytokine production (Fig. 1EGo). These results suggest that the activation of peritoneal exudate macrophages was unrelated to the solubility of the water-insoluble {alpha}-glucan preparation.


Figure 1
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  		Figure 1. Cell proliferation and cytokine production by mouse immune cells following stimulation with water-insoluble {alpha}-glucans. (A) Mouse splenocytes (4 x 105 cells in 200 µL of complete RPMI 1640 medium) were stimulated for 4 days with the indicated concentrations of the test materials indicated in the panel, after which cell proliferation was determined by 3H-thymidine incorporation. (B,C,D) Mouse peritoneal exudate macrophages (5 x 105 cells/mL of complete RPMI 1640 medium) were stimulated for 24 hrs with increasing concentrations of the test materials, after which the production of TNF-{alpha} (B), IL-6 (C), and IL-1β (D) was assessed with the use of cytokine ELISA kits. Results represent the means ± SE from 9 wells (3 experiments performed in triplicate). *p < 0.01, compared with sucrose-stimulated cells; **p < 0.01, compared with dextran T-2000-stimulated cells; ***p < 0.01, compared with water-soluble {alpha}-glucan from S. sobrinus-stimulated cells; ****p < 0.01, compared with muramyl dipeptide-stimulated cells. *****p < 0.01, compared with S. sobrinus cell wall extracts-stimulated cells. MDP ({blacktriangledown}); muramyl dipeptide, CW ({square}); S. sobrinus cell wall extracts containing peptidoglycan and lipoteichoic acid, WIG ({blacksquare}); water-insoluble {alpha}-glucans produced by S. sobrinus GTFs, WSG ({triangledown}); water-soluble {alpha}-glucan produced by S. sobrinus GTFs, Dextran ({blacktriangleup}); dextran T-2000; •, LPS; {triangleup}, sucrose. (E) Effects of NaOH-treated water-insoluble {alpha}-glucans on cytokine production by peritoneal exudate macrophages. Water-insoluble {alpha}-glucans (3, 10, 30, or 100 µg) were dissolved in 1 N of NaOH, and diluted 1000-fold with complete RPMI 1640 medium, after which 1 mL of each solution (3, 10, 30, or 100 µg/mL of water-insoluble {alpha}-glucans) was added to 5 x 105 cells of peritoneal exudate macrophages. TNF-{alpha} and IL-6 production was assessed with the use of cytokine ELISA kits. Results represent the means ± SE from 9 wells (3 experiments performed in triplicate).

 
Activation of Peritoneal Exudate Macrophages by Water-insoluble {alpha}-glucans Was Not Due to Contamination by LPS, Peptidoglycan, β-glucan, or Water-soluble {alpha}-glucans
We found that slight contamination by endotoxin (< 10 pg/100 µg of water-insoluble {alpha}-glucans), peptidoglycan (< 2.5 ng/100 µg of water-insoluble {alpha}-glucans), and β-glucans (< 25 pg/100 µg of water-insoluble {alpha}-glucans) occurred in our experiments. Since 10 pg/mL of LPS, 2.5 ng/mL of peptidoglycan, or 25 pg/mL of β-glucans could not activate macrophages to produce TNF-{alpha} (less than 78 pg/mL) or enhance TNF-{alpha} production by water-insoluble {alpha}-glucan-stimulated macrophages (Fig. 2AGo; APPENDIX 5), we concluded that the observed responses to water-insoluble {alpha}-glucans were not due to contamination by those agents. We also confirmed that dextran T-2000, water-insoluble {alpha}-glucans, and water-soluble {alpha}-glucans each induced TNF-{alpha} production by J774.1 cells, while a lower level of TNF-{alpha} was produced by peritoneal exudate macrophages when stimulated with either dextran T-2000 or water-soluble {alpha}-glucans, as compared with water-insoluble {alpha}-glucans (Fig. 2BGo; APPENDIX 6).


Figure 2
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  		Figure 2. Roles of LPS, peptidoglycan, β-glucan, and water-soluble {alpha}-glucans in the activation of peritoneal exudate macrophages by water-insoluble {alpha}-glucans. (A) 10 pg/mL of LPS, 2.5 ng/mL of type III peptidoglycan, 25 pg/mL of β-1,3-glucan, 30 µg/mL of water-insoluble {alpha}-glucans with 10 pg/mL of LPS, 30 µg/mL of water-insoluble {alpha}-glucans with 2.5 ng/mL of type III peptidoglycan, 30 µg/mL of water-insoluble {alpha}-glucans with 25 pg/mL of β-1,3-glucan, or 30 and 100 µg/mL of water-insoluble {alpha}-glucans was added to 5 x 105 cells of peritoneal exudates macrophages for 24 hrs. WIG, water-insoluble {alpha}-glucans; PGN, peptidoglycan. (B) J774.1 cells (105 cells/mL of complete RPMI 1640 medium) or peritoneal exudate macrophages (5 x 105 cells/mL of complete RPMI 1640 medium) were stimulated with water-insoluble {alpha}-glucans (WIG; {blacksquare}), water-soluble {alpha}-glucans from S. sobrinus (WSG; {triangledown}), dextran T-2000 (Dextran; {blacktriangleup}), or sucrose ({triangleup}) at the indicated concentrations for 24 hrs, after which the production of TNF-{alpha} was assessed by means of a cytokine ELISA kit. Results represent the means ± SE from 9 wells (3 experiments performed in triplicate). *p < 0.01, compared with cells stimulated with WIG; **p < 0.01, compared with sucrose-stimulated cells. ND; not detectable (less than 78 pg/mL).

 
TLR2, 4-, NOD1-, and NOD2-independent Cell Activation by Stimulation with Water-insoluble {alpha}-glucans
Since complement receptor 3, a scavenger receptor, TLR, and/or NOD modulate innate immunity in mouse macrophages, we attempted to determine which molecules involved in the activation of macrophages by stimulation with water-insoluble {alpha}-glucans were modulated, using utilized RNA interference assays targeting TLR2, TLR4, NOD1, and NOD2. The targeted sequences are listed in Fig. 3AGo. The mRNA levels of the 4 molecules were suppressed by specific siRNA in J774.1 cells in 24-hour cultures (Fig. 3BGo). However, their suppression did not decrease the level of TNF-{alpha} production following stimulation with water-insoluble {alpha}-glucans (Fig. 3CGo).


Figure 3
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  		Figure 3. Effect of macrophage activation stimulated with water-insoluble {alpha}-glucans on suppression of TLR2, TLR4, NOD1, and NOD2 mRNA expression. (A) The primers used in the RT-PCR assay and sequence of the target mRNA for TLR2, TLR4, NOD1, and NOD 2. (B) J774.1 cells were transfected with siRNA of TLR2, TLR4, NOD1, NOD2, or buffer only with the use of TransIT-TKO transfection reagent. Total RNA was extracted 24 hrs after transfection, and RT-PCR was performed. (C) J774.1 or transfected J774.1 cells (105 cells/mL) were stimulated with 1 mg/mL of water-insoluble {alpha}-glucan for 24 hrs, and the TNF-{alpha} in the culture supernatants was measured by a TNF-{alpha} ELISA kit.

 
Stimulation of Human Monocytes and Polymorphonuclear Cells, and Induction of Chemotaxis and H2O2 Production by Water-insoluble {alpha}-glucans
There are few studies of the effects of {alpha}-glucans on cytokine production in human monocytes. In the present study, we attempted to determine whether water-insoluble {alpha}-glucans induce cytokine production in human peripheral monocytes. A high level of TNF-{alpha} was detected in the supernatants of water-insoluble {alpha}-glucan-stimulated monocytes (1789 ± 445 pg/mL at 1 mg/mL; Fig. 4AGo). We also found that IL-8 was produced by monocytes stimulated with 0.1 to 10 µg/mL of water-insoluble {alpha}-glucans (APPENDIX 7). Furthermore, cytokine production by peritoneal exudate macrophages stimulated with water-insoluble {alpha}-glucans was significantly higher (p < 0.05) than when those were stimulated with muramyl dipeptide, water-soluble {alpha}-glucans, or dextran T-2000 (Figs. 4A, 4BGo; APPENDIX 8).


Figure 4
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  		Figure 4. Cytokine production in human monocytes induced by water-insoluble {alpha}-glucans and chemotaxis and H2O2 production in human polymorphonuclear cells, following stimulation with water-insoluble {alpha}-glucans. (A,B) Human monocytes (5 x 105 cells/mL of complete RPMI 1640 medium) were stimulated for 48 hrs with increasing concentrations of water-insoluble {alpha}-glucans (WIG; {blacksquare}), water-soluble {alpha}-glucans from S. sobrinus (WSG; {triangledown}), S. sobrinus cell wall extracts including peptidoglycan and lipoteichoic acid (CW; {square}), dextran T-2000 (Dextran; {blacktriangleup}), muramyl dipeptide (MDP; {blacktriangledown}), LPS (•), or sucrose ({triangleup}), after which the production of TNF-{alpha} (A) and IL-8 (B) was assessed with the use of cytokine ELISA kits. Results represent means ± SE from 12 wells (4 experiments performed in triplicate). *p < 0.01, compared with sucrose-stimulated cells; **p < 0.01, compared with dextran T-2000-stimulated cells; ***p < 0.01, compared with water-soluble {alpha}-glucan from S. sobrinus-stimulated cells; ****p < 0.01, compared with muramyl dipeptide-stimulated cells. *****p < 0.01, compared with S. sobrinus cell wall extract-stimulated cells. (C) Polymorphonuclear cells (5 x 106 cells/mL of complete RPMI 1640 medium) were labeled with BCECF-AM, and chemotaxis assays were performed with the indicated concentrations of water-insoluble {alpha}-glucans (WIG) and water-soluble {alpha}-glucans (WSG). Chemotaxis of polymorphonuclear cells was measured following a 24-hour treatment with the supernatant from monocytes stimulated with the indicated concentrations of sucrose (light-gray columns), water-insoluble {alpha}-glucans (black columns), or water-soluble {alpha}-glucans from S. sobrinus (dark-gray columns). (D) Polymorphonuclear cells (5 x 106 cells/mL of complete RPMI 1640 medium) were pre-treated with C5a, sucrose, water-insoluble {alpha}-glucans (WIG), or water-soluble {alpha}-glucans from S. sobrinus (WSG) for 1 hr at 37°C. Following pre-treatment, phorbol 12-myristate 13-acetate and DCFH-DA were added to the cell suspensions. After a one-hour incubation at 37°C, the increase in total fluorescence was determined. Results represent the means ± SE from 15 wells (5 experiments performed in triplicate). #p < 0.01, compared with polymorphonuclear cells stimulated with the supernatant from PBS-stimulated human monocytes; ##p < 0.01, compared with polymorphonuclear cells stimulated with PBS.

 
IL-8 is known to promote the chemotaxis of human polymorphonuclear cells and memory T-cells, and we found that the culture supernatants from human monocytes stimulated with water-insoluble {alpha}-glucans, but not sucrose, induced chemotaxis of human polymorphonuclear cells (Fig. 4CGo; APPENDIX 9). Although the culture supernatants of water-soluble {alpha}-glucan-stimulated human monocytes induced chemotaxis in human polymorphonuclear cells, we did not detect IL-8 in the supernatants. Since factors other than IL-8, including Gro{alpha}, Groβ, ENA-78, GCP-2, and NAO-2, can induce chemotaxis of polymorphonuclear cells, we speculated that production of chemokines other than IL-8 mediates the ability of water-soluble {alpha}-glucans to promote chemotaxis. Since H2O2 released by polymorphonuclear cells is an important mediator of endothelial cell injury and vascular inflammation (Lounsbury et al., 2000), we also examined whether water-insoluble {alpha}-glucan-stimulated polymorpho-nuclear cells produced H2O2. Water-insoluble {alpha}-glucans, but not water-soluble {alpha}-glucans, induced the production of H2O2 in human polymorphonuclear cells (Fig. 4DGo; APPENDIX 10), suggesting that water-insoluble {alpha}-glucans induce chemotaxis and inflammatory responses in polymorphonuclear cells.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, water-insoluble {alpha}-glucans synthesized by S. sobrinus GTF-I, but not sucrose, activated mouse peritoneal exudate macrophages to produce pro-inflammatory cytokines. Activation of macrophages by water-insoluble {alpha}-glucans was revealed in experiments with both the insoluble and soluble forms. We also concluded that the effects of the water-insoluble {alpha}-glucan preparations were not due to contamination with LPS, peptidoglycan, β-glucan, or sucrose. Macrophage activation by stimulation with water-insoluble {alpha}-glucans was not suppressed by depletion of TLR2, TLR4, NOD1, or NOD2, suggesting that the stimulatory pathway of water-insoluble {alpha}-glucans is different from that of LPS (TLR4 pathway), peptidoglycan (TLR2 pathway), lipoteichoic acid (TLR2 pathway), and muramyl dipeptide (NOD1 pathway). Furthermore, we also observed that the water-insoluble {alpha}-glucan preparation activated human monocytes and polymorphonuclear cells, suggesting that {alpha}-glucans have an ability to modulate the human immune system.

Glucans synthesized by S. sobrinus contain {alpha}-1,3-glucosyl linkages (mutan) and {alpha}-1,6-glucosyl linkages (dextran) (Hanada et al., 2002). Water-soluble {alpha}-glucans contain glucose primarily with {alpha}-1,6-glucosyl linkages, whereas water-insoluble {alpha}-glucans possess a high degree of branching involving {alpha}-1,3- glucosyl linkages (Schuster, 1983). In the present study, much lower levels of TNF-{alpha}, IL-8, and H 2O 2 were produced by monocytes/macrophages stimulated with water-soluble {alpha}-glucans than with water-insoluble {alpha}-glucans, whereas the 2 types of {alpha}-glucans had nearly the same effects on the production of TNF-{alpha} in J774.1 cells. These results suggest that the immunological properties of polysaccharides with {alpha}-1,3-glucosyl linkages are different from those with {alpha}-1,6 glucosyl linkages.

Water-insoluble {alpha}-glucans induce innate immune responses in monocytes/macrophages; however, the mechanism for the activation of monocytes/macrophages and polymorphonuclear cells to induce inflammatory immune responses remains unclear. Previous studies have shown that macrophage activation by zymosan, a fungal β-glucan, is due to 2 signaling pathways, a pathway in collaboration with TLR-2, 6, and CD14, and a Dectin-1-dependent pathway (Underhill et al., 1999; Ozinsky et al., 2000; Brown et al., 2002). Inflammatory responses triggered by zymosan are linked to its phagocytosis, a process that is mediated by Dectin-1, but not TLRs (Frasnelli et al., 2005). Furthermore, Dectin-1 is a major receptor for both non-opsonized-particulates and soluble β-glucans on macrophages (Brown et al., 2002). In the present study, activation of peritoneal exudate macrophages was found to be unrelated to the solubility of the water-insoluble {alpha}-glucan preparation, while TLR2 was weakly associated with activation of macrophages by water-insoluble {alpha}-glucans. We speculate that activation of cells by the {alpha}-glucans is associated with a Dectin-1 dependent pathway.

Periodontal diseases are typical inflammatory diseases that occur in the oral cavity (Schuster, 1983), and include inflammation of gingival tissues and the accumulation of dental plaque in the coronal pockets. Investigators have speculated that several different strains of bacteria (e.g., Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, and Bacteroides forsythus) in dental plaque invade oral squamous cells and induce inflammation in gingival tissues (Schuster, 1983; Van Dyke and Serhan, 2003). Since oral squamous tissues hinder the contact of water-insoluble {alpha}-glucans with monocytes/macrophages, polymorphonuclear cells, and lymphocytes in the lamina propria and distinct submucous layer, it is difficult for water-insoluble {alpha}-glucans to induce an inflammatory immune response in the gingiva. However, dental plaque contains abundant bacteria that injure oral squamous cells, and the cytotoxicity of bacteria toward squamous cells may allow for water-insoluble {alpha}-glucans to reach monocytes/macrophages and polymorphonuclear cells, resulting in inflammatory responses in the gingiva. Therefore, we speculate that water-insoluble {alpha}-glucans help mediate the induction of periodontal diseases.

In summary, we found that water-insoluble {alpha}-glucans produced by S. sobrinus activate macrophages and polymorphonuclear cells in mice and humans, resulting in the induction of inflammatory immune responses. Therefore, we propose that water-insoluble {alpha}-glucans participate in the pathogenicity of dental caries as well as inflammatory diseases in the oral cavity.


   ACKNOWLEDGMENTS
 
This research was supported by Grants-in-Aid for Scientific Research (B)-(2) and (C)-(2) from the Japan Society for the promotion of Science, and by a Grant-in-Aid for Scientific Research on Priority Areas and the 21st Century COE Program of the Ministry of Education, Culture, Sports, Science, and Technology of Japan.


   FOOTNOTES
 
A supplemental appendix to this article is published electronically only at http://www.dentalresearch.org.

Received for publication December 28, 2005. Revision received October 25, 2006. Accepted for publication November 7, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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Journal of Dental Research, Vol. 86, No. 3, 242-248 (2007)
DOI: 10.1177/154405910708600309
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