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Infection and Immunity, October 2003, p. 5598-5604, Vol. 71, No. 10
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.10.5598-5604.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Augmentation of Actinobacillus actinomycetemcomitans Invasion of Human Oral Epithelial Cells and Up-Regulation of Interleukin-8 Production by Saliva CD14

Atsuko Takayama,^1,2,3 Aya Satoh,¹ Tomoko Ngai,¹ Takashi Nishimura,¹ Keiji Ikawa,^1,2,3 Takami Matsuyama,⁴ Hidetoshi Shimauchi,² Haruhiko Takada,³ and Shunji Sugawara^1*

Division of Oral Immunology,¹ Division of Periodontology and Endodontology,² Division of Oral Microbiology, Department of Oral Biology, Tohoku University Graduate School of Dentistry, Sendai 980-8575,³ Department of Immunology and Medical Zoology, Kagoshima University Graduate School of Medicine, Kagoshima 890-8544, Japan⁴

Received 28 May 2003/ Returned for modification 8 July 2003/ Accepted 21 July 2003

Top Abstract Introduction Materials and Methods Results Discussion References

 
It has recently been shown that human salivary glands constitutively express CD14, an important molecule in innate immunity, and that a soluble form of CD14 is secreted in saliva. The concentration of CD14 in parotid (a serous gland) saliva was comparable to that in normal serum and 10-fold the amount in whole saliva, although the physiological function of saliva CD14 remained unclear. Actinobacillus actinomycetemcomitans is a periodontopathic bacterium and is able to invade oral epithelial cells. The present study showed that upon exposure to live A. actinomycetemcomitans Y4 for 2 h, human oral epithelial HSC-2 cells produced interleukin-8 (IL-8) for a further 24 h and whole saliva augmented the production induced by A. actinomycetemcomitans Y4. Parotid saliva showed a more pronounced effect on the production of IL-8 than whole saliva. Neither saliva preparation itself had IL-8-inducing activity. Parotid saliva exhibited antibacterial activity against a low concentration of A. actinomycetemcomitans Y4, but recombinant CD14 did not show the activity. The internalization of A. actinomycetemcomitans Y4 into HSC-2 cells was inhibited by cytochalasin B, indicating that the process was actin dependent, and depletion of CD14 from parotid saliva inhibited the invasion and, as a consequence, inhibited production of IL-8. Furthermore, human recombinant CD14 augmented invasion and IL-8 production. These results suggest that saliva CD14 promoted the invasion of oral epithelial cells by A. actinomycetemcomitans and consequently augmented the production of IL-8, playing an important role in innate immunity in the oral cavity.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Oral epithelial cells are the first cells encountered by bacteria in the periodontal tissues. In addition to acting as a physical barrier against invasion by pathogenic organisms, oral (gingival) epithelial cells are able to produce a wide variety of cytokines and chemokines (18, 31, 32, 34, 35, 40) and to express intercellular adhesion molecule-1 on the cell surface (13, 34), implying that the cells actively participate in host defense mechanisms. Actinobacillus actinomycetemcomitans, a gram-negative facultative capnophilic rod, has been implicated as a causative agent of aggressive and chronic periodontitis (21, 37). A. actinomycetemcomitans is able to invade oral epithelial cells, indicating that the process is important for the establishment of the diseases (22). On the other hand, challenge with A. actinomycetemcomitans induces oral epithelial cells to produce interleukin-8 (IL-8) and to express intercellular adhesion molecule-1 (12, 13). This finding indicates that oral epithelial cells play an important role in the initiation of the innate immune response in the periodontal tissues, contributing to the infiltration and activation of neutrophils at the site of bacterial infection.

CD14 is a 55-kDa glycosylphosphatidylinositol-anchored glycoprotein that is expressed mainly on the surface of monocytes and macrophages (11). CD14 functions as a bacterial pattern recognition receptor for lipopolysaccharide (LPS) and many bacterial components in the innate immune response to bacterial invasion (25, 38). Recently, a family of Toll-like receptors (TLRs) was found to be essential for microbial recognition and signaling in innate immunity (1), and CD14 mediates sensitive responses to LPS by facilitating interaction with TLR4 in association with MD-2, an adapter molecule of TLR-4 (1). CD14 also exists in serum (4) and milk (7, 17) in a soluble form (sCD14). It is reported that sCD14 in serum decreases cellular responses to LPS by transferring cell-bound LPS to serum lipoproteins and lactoferrin (3, 16, 21) and that sCD14 at low concentrations mediates the activation of CD14-negative cells, such as endothelial and epithelial cells, by LPS (8, 26). CD14 also contributes to the phagocytosis of gram-negative bacteria, including A. actinomycetemcomitans, by monocytes and monocytic cells (9, 24, 28).

Saliva, a complex mix of fluids from major (parotid, submandibular, and sublingual) and minor salivary glands, is a valuable oral fluid that is critical to the maintenance of oral health, including the health of the oral mucous membrane and the teeth (20). Saliva contains a number of antimicrobial agents, secretory immunoglobulin A (secretory IgA), proteins (glycoproteins, statherins, agglutinins, histidine-rich proteins, and proline-rich proteins), mucins, lactoferrin, enzymes (lysozyme and peroxidase), and antimicrobial peptides (14, 33). The concerted action of these agents is thought to provide a multifunctional protective network against microorganisms. In addition, it has recently been shown that major salivary glands constitutively express CD14 and that sCD14 is secreted in saliva (36). The concentration of CD14 in parotid (a serous gland) saliva was comparable to that in normal serum and 10-fold the amount in whole saliva. In contrast, levels of LPS-binding protein (LBP; the serum protein that accelerates the binding of LPS to CD14 [10]) in whole and parotid saliva were below the detectable limit.

The physiological function of saliva CD14 remains unclear, and the evidence that monocytes phagocytose gram-negative bacteria through a CD14-dependent mechanism (9, 24, 28) led us to investigate whether saliva CD14 is involved in the invasion and activation of oral epithelial cells by periodontopathic bacteria. The present study showed that saliva CD14 augmented the invasion of oral epithelial cells by A. actinomycetemcomitans in culture and, as a consequence, up-regulated production of IL-8 by the epithelial cells. The underlying mechanism is discussed below.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial strain and reagents. A. actinomycetemcomitans strain Y4 was kindly provided by T. Nishihara (Kyushu Dental College, Kitakyushu, Japan) and grown in brain heart infusion (Difco Laboratories, Detroit, Mich.) at 37°C in a 5% CO₂ atmosphere in air (24). Solid medium was prepared by adding agar (Difco) to the liquid medium to a final concentration of 1.5% (wt/vol). Human recombinant CD14 (rCD14) was obtained from Biometec (Greifswald, Germany). All other reagents were obtained from Sigma-Aldrich (St. Louis, Mo.) unless otherwise indicated.

Cells and cell culture. The human oral epithelial cell lines HSC-2 (23) and KB (6), established from squamous cell carcinomas, were obtained from the Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University (Sendai, Japan). HSC-2 was grown in RPMI 1640 medium with heat-inactivated fetal calf serum (FCS) (Life Technologies, Grand Island, N.Y.), and KB was grown in alpha minimal essential medium (Life Technologies) with 10% FCS (31, 35).

Collection of saliva. Whole saliva was collected from healthy adult donors, aged 22 to 24 years, into sterile plastic tubes after the collection of parotid saliva. Parotid saliva was collected with the aid of Schaefer cups placed over the Stensen's duct (27). The saliva samples were immediately clarified by centrifugation at 14,000 x g for 5 min at 4°C and passed through sterile membrane filters (0.22-µm pore size). Clarified saliva samples were immediately used or aliquoted and frozen at -70°C until use. Saliva preparations were diluted in sterile phosphate-buffered saline. The experimental procedures were approved by the Ethical Review Board of Tohoku University Graduate School of Dentistry (Sendai, Japan).

Antibacterial effect of saliva. Live A. actinomycetemcomitans Y4 cells at various doses were incubated with saliva or rCD14 for a given period and then plated onto agar. After the incubation at 37°C in a 5% CO₂ atmosphere for 48 h, CFU were enumerated. Each sample was assayed in triplicate.

Coculture of oral epithelial cells with A. actinomycetemcomitans. The coculture of oral epithelial cells with A. actinomycetemcomitans was conducted as described previously (12, 13) with minor modification. HSC-2 cells (10⁵ cells/well) were seeded in RPMI 1640 medium with 10% FCS in wells of 24-well tissue culture plates (Falcon; BD Labware, Franklin Lakes, N.J.). After incubation for 1 day at 37°C in a 5% CO₂ atmosphere, confluent monolayers of HSC-2 cells were washed with antibiotic-free RPMI 1640 medium three times, and then live A. actinomycetemcomitans Y4 cells were added at various doses in 200 µl of antibiotic-free medium without serum and the mixture was centrifuged at 900 x g for 5 min at room temperature. The coculture was incubated at 37°C for 2 h to allow interactions between bacteria and HSC-2 cells in the presence or absence of saliva samples or rCD14. Monolayers were then washed three times, and the cultures were further incubated for 24 h in the medium with 10% FCS and 0.1 mg of gentamicin (12)/ml. After the incubation, the supernatants were collected, and the levels of IL-8 produced by HSC-2 cells in the supernatants were determined with an OptEIA human IL-8 enzyme-linked immunosorbent assay (ELISA) kit (BD Pharmingen, San Diego, Calif.). The concentrations of IL-8 in the culture supernatants were determined with the Softmax data analysis program (Molecular Devices, Menlo Park, Calif.). Each sample was assayed in triplicate.

To enumerate CFU of internalized bacteria, the 2-h coculture was washed three times and further incubated in the medium with 0.1 mg of gentamicin/ml for 2 h to kill extracellular bacteria (22). Monolayers were then lysed in 1 ml of distilled water and plated onto agar.

Depletion of CD14 from saliva. Anti-human CD14 monoclonal antibody (MAb) D10 (mouse IgG1) (39) was purified from ascitic fluid as described previously (30). The purified D10 and control MAb were coupled to HiTrap N-hydroxysuccinimide-activated Sepharose columns (Amersham Biosciences, Piscataway, N.J.) to yield 1 mg of IgG/ml of packed gel, in accordance with the manufacturer's protocol. One milliliter of parotid saliva was applied to the column for 1 h at room temperature, eluted with 1 ml of phosphate-buffered saline, and then passed through a sterile membrane filter (0.22-µm pore size). Levels of CD14 in the saliva preparations were measured with a human sCD14 ELISA kit (BioSource Europe, Fleurus, Belgium).

Statistical analysis. All of the experiments in this study were conducted at least three times to confirm the reproducibility of the results. Experimental values are given as means ± standard deviations (SD) of results from triplicate assays. The statistical significance of differences between two means was evaluated by using a one-way analysis of variance with the Bonferroni or Dunnett method, and P values of less than 0.05 were considered significant.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Involvement of human saliva in production of IL-8 by oral epithelial cells activated by A. actinomycetemcomitans. We first examined whether human saliva is involved in the production of IL-8 by human oral epithelial cells activated by a periodontopathic bacterium, A. actinomycetemcomitans. Exposure to live A. actinomycetemcomitans Y4 at a concentration of 10⁸ CFU/well for 2 h induced human oral epithelial HSC-2 cells to produce IL-8, which was detected in supernatants from subsequent 24-h cultures, and the presence of whole saliva at a 50% concentration further augmented the production (Fig. 1A). The presence of parotid saliva at a 50% concentration during the bacterial challenge had a more pronounced effect on the production of IL-8 than the presence of whole saliva (Fig. 1B). The two saliva preparations themselves had no such activity, and the presence of the preparations at 100% concentrations reduced the basal and induced levels of IL-8 production. The reduction was probably due to damage of the HSC-2 cells caused by 100% saliva because the cell viability was reduced to less than 10% upon 24-h incubation after challenge with A. actinomycetemcomitans Y4 and 100% whole or parotid saliva for 2 h. Taking into account the results shown in Fig. 1, we used parotid saliva to elucidate the underlying mechanism.

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FIG. 1. Effect of saliva on A. actinomycetemcomitans-induced IL-8 production by oral epithelial cells. Confluent HSC-2 cells were cocultured with A. actinomycetemcomitans Y4 (A.a; 108 CFU/well) in the presence or absence of whole (A) and parotid (B) saliva at the indicated concentrations for 2 h at 37°C. Monolayers were washed and further incubated for 24 h with 0.1 mg of gentamicin/ml. Concentrations of IL-8 in the culture supernatants were determined by ELISA. The results are expressed as the means ± SD of results for triplicate cultures. **, P is <0.01 compared with the saliva-free control. The experiments were carried out with saliva samples from three donors, and the results presented are representative of similar results from three different experiments.

Live A. actinomycetemcomitans Y4 showed more IL-8-inducing activity at 10⁹ CFU/well than at 10⁸ CFU/well, and parotid saliva had no significant effect on production (Fig. 2). In addition, live A. actinomycetemcomitans Y4 at 10⁷ CFU/well did not affect the production of IL-8 and parotid saliva did not alter the level of IL-8 (data not shown). HSC-2 cells produced IL-8 in substantial amounts in response to A. actinomycetemcomitans Y4, which was consistent with observations with primary gingival epithelial cells (12). Although KB cells were utilized for the invasion experiment as an in vitro system (22), they did not produce IL-8 in response to live A. actinomycetemcomitans Y4 (data not shown). Therefore, HSC-2 cells were used for further experiments in vitro.

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FIG. 2. Effect of concentrations of A. actinomycetemcomitans on the saliva-mediated augmentation of IL-8 production by oral epithelial cells. Confluent HSC-2 cells were cocultured with A. actinomycetemcomitans Y4 (A.a; 108 or 109 CFU/well) in the presence or absence of parotid saliva at the indicated concentrations for 2 h at 37°C. Monolayers were washed and further incubated for 24 h with 0.1 mg of gentamicin/ml. Concentrations of IL-8 in the culture supernatants were determined by ELISA. The results are expressed as the means ± SD of results for triplicate cultures. **, P is <0.01 compared with the saliva-free control. The experiments were carried out with saliva samples from three donors, and the results presented are representative of similar results from three different experiments.

Activity of parotid saliva against A. actinomycetemcomitans. We next examined the activity of parotid saliva against A. actinomycetemcomitans. Incubation of live A. actinomycetemcomitans Y4 cells at 10⁷ CFU/ml with 50% parotid saliva for 2 h significantly reduced the number of CFU to 6.5% of the original dose, with further reduction occurring with 100% saliva (Fig. 3A). Live A. actinomycetemcomitans Y4 cells at 10⁸ CFU/ml were relatively resistant, and the number of CFU was reduced to 12.8% of the original dose when the bacteria were incubated with 100% parotid saliva. Parotid saliva showed no activity against live A. actinomycetemcomitans Y4 at a concentration of 10⁹ CFU/ml. Parotid saliva contains CD14, one of the most important molecules involved in innate immune recognition (1), at the same levels that serum does (36); therefore, we next examined whether rCD14 exhibits activity against live A. actinomycetemcomitans Y4 at 10⁷ CFU/ml. Figure 3B shows that rCD14 did not reduce the number of CFU even at 10 µg/ml, which is a higher concentration than that found in serum or parotid saliva. The results indicate that the antibacterial activity of saliva is limited, that parotid saliva is active against periodontopathic A. actinomycetemcomitans only at low concentrations of the bacterium and has no effect on the organism at high concentrations, and that saliva CD14 itself does not contribute to the antibacterial activity.

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FIG. 3. Activity of parotid saliva and CD14 against A. actinomycetemcomitans. (A) Live A. actinomycetemcomitans Y4 (A.a) was incubated at the indicated concentrations with the given concentrations of parotid saliva for 2 h and plated onto agar, and then CFU were enumerated. (B) Live A. actinomycetemcomitans Y4 at a concentration of 107 CFU/ml was incubated with the indicated concentrations of human rCD14 for 2 and 4 h and plated onto agar. CFU were then enumerated. The results are expressed as the means ± SD of results for triplicate cultures. **, P is <0.01 compared with the saliva-free control. The experiments were carried out with saliva samples from three donors, and the results presented are representative of similar results from three different experiments.

Saliva CD14-dependent invasion of oral epithelial cells by A. actinomycetemcomitans. Previous reports showed that A. actinomycetemcomitans invades oral epithelial cells (22) and induces them to produce IL-8 (12, 13) and that monocytes phagocytose gram-negative bacteria, including A. actinomycetemcomitans, through a CD14-dependent mechanism (9, 24, 28). Therefore, we next investigated whether CD14 in saliva is involved in the invasion and activation of oral epithelial cells by A. actinomycetemcomitans. Figure 4 shows that parotid saliva at a 50% concentration significantly increased the number of internalized A. actinomycetemcomitans Y4 cells in HSC-2 cells and that preincubation with cytochalasin B, an inhibitor of actin polymerization (19), at 30 and 60 nM significantly inhibited the internalization. Vehicle control (dimethyl sulfoxide [DMSO]) had no effect on the internalization. The results clearly indicate that the invasion by A. actinomycetemcomitans Y4 requires the cytoskeletal movement of oral epithelial cells, as described previously (5).

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FIG. 4. Effect of cytochalasin B on invasion of oral epithelial cells by A. actinomycetemcomitans. Confluent HSC-2 cells were preincubated with cytochalasin B (Cyto B) at the indicated concentrations or with the respective vehicle control (DMSO) for 30 min. Monolayers were then cocultured with live A. actinomycetemcomitans Y4 (108 CFU/well) in the presence or absence of 50% parotid saliva, cytochalasin B, or vehicle control (DMSO) at the dose indicated for 2 h. The cocultures were washed three times and further incubated in the medium with 0.1 mg of gentamicin/ml for 2 h. Monolayers were then lysed in distilled water and plated onto agar, and CFU were enumerated. The results are expressed as the means ± SD of results for triplicate cultures. **, P is <0.01 compared with the respective control. The experiments were carried out with saliva samples from three donors, and the results presented are representative of similar results from three different experiments. +, present; -, absent.

We then examined whether CD14 in parotid saliva is involved in augmenting the invasion. The depletion of CD14 from parotid saliva by using an anti-CD14 MAb affinity column significantly decreased numbers of A. actinomycetemcomitans Y4 cells in HSC-2 cells compared those in HSC-2 cells incubated with untreated parotid saliva (Fig. 5A). The control parotid saliva preparation obtained by using an irrelevant MAb was comparable to untreated parotid saliva. Concomitantly, the augmentation by parotid saliva of A. actinomycetemcomitans Y4-induced IL-8 production by HSC-2 cells was significantly decreased by depletion of CD14 from the saliva (Fig. 5B).

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FIG. 5. Effect of CD14 depletion from saliva on invasion and activation of oral epithelial cells by A. actinomycetemcomitans. (A) Confluent HSC-2 cells were cocultured with live A. actinomycetemcomitans Y4 (A.a) (108 CFU/well) in the presence or absence of parotid saliva, CD14-depleted by using anti-CD14 (CD14-) or control parotid saliva obtained by using the control IgG affinity column (control) (each at a 50% concentration) for 2 h. The cocultures were washed three times and further incubated in the medium with 0.1 mg of gentamicin/ml for 2 h. Monolayers were then lysed in distilled water and plated onto agar. CFU were then enumerated. (B) Confluent HSC-2 cells were cocultured with live A. actinomycetemcomitans Y4 (108 CFU/well) in the presence or absence of parotid saliva, parotid saliva depleted of CD14 (CD14-), or control parotid saliva control (each at a 50% concentration) for 2 h. Monolayers were washed and further incubated for 24 h with 0.1 mg of gentamicin/ml. Concentrations of IL-8 in the culture supernatants were determined by ELISA. The results are expressed as the means ± SD of results for triplicate cultures. *, P is <0.05, and **, P is <0.01 compared with normal saliva. The experiments were carried out with saliva samples from three donors, and the results presented are representative of similar results from three different experiments.

Finally, we examined whether addition of CD14 promotes invasion and the production of IL-8. Human rCD14 at 5 µg/ml augmented the invasion by A. actinomycetemcomitans Y4 of HSC-2 cells, comparable to the effect of 50% parotid saliva (Fig. 6A). Human rCD14 at 5 µg/ml also augmented the production of IL-8 induced by A. actinomycetemcomitans Y4 at a concentration of 10⁸ CFU/well but not that induced by the organism at a concentration of 10⁹ CFU/well (Fig. 6B), which was the case for parotid saliva (Fig. 2).

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FIG. 6. Effect of rCD14 on invasion and activation of oral epithelial cells by A. actinomycetemcomitans. (A) Confluent HSC-2 cells were cocultured with live A. actinomycetemcomitans Y4 (108 CFU/well) in the presence or absence of parotid saliva (50%) or rCD14 (5 µg/ml) for 2 h. The cocultures were washed three times and further incubated in the medium with 0.1 mg of gentamicin/ml for 2 h. Monolayers were then lysed in distilled water and plated onto agar, and CFU were enumerated. +, present; -, absent. (B) Confluent HSC-2 cells were cocultured with live A. actinomycetemcomitans Y4 (108 or 109 CFU/well) in the presence or absence of rCD14 at the indicated concentrations for 2 h. Monolayers were washed and further incubated for 24 h with 0.1 mg of gentamicin/ml. Concentrations of IL-8 in the culture supernatants were determined by ELISA. The results are expressed as the means ± SD of results for triplicate cultures. *, P is <0.05, and **, P is <0.01 compared with the control (without saliva or rCD14). The experiments were carried out with saliva samples from three donors, and the results presented are representative of similar results from three different experiments.

Top Abstract Introduction Materials and Methods Results Discussion References

 
It has recently been shown that human salivary glands constitutively express CD14, one of the most important molecules involved in innate immunity, and that sCD14 is secreted in saliva (36). The concentration of CD14 in parotid (a serous gland) saliva (1 to 2 µg/ml) was comparable to that in normal serum and 10-fold the amount in whole saliva (36), although the physiological function of CD14 in the oral cavity remained unclear. The present study clearly showed that saliva CD14 promoted the invasion of oral epithelial cells by periodontopathic A. actinomycetemcomitans and consequently augmented the production of IL-8 by oral epithelial cells. Parotid saliva had a more pronounced effect on the augmentation of A. actinomycetemcomitans-induced IL-8 production than whole saliva. The result is probably due to the difference in the amounts of CD14 in the saliva samples (36).

One group previously used KB cells to show that A. actinomycetemcomitans invades oral epithelial cells because KB cells are most susceptible to invasion by A. actinomycetemcomitans (22). Another group used primary normal human oral keratinocytes and immortalized cell lines to show that treatment with A. actinomycetemcomitans for 2 h induces oral epithelial cells to produce IL-8 (12, 13). The present study clearly showed that A. actinomycetemcomitans Y4 invaded oral epithelial cells and subsequently induced production of IL-8. HSC-2 cells were mainly used in this study because KB cells do not produce IL-8 after treatment with A. actinomycetemcomitans Y4, although the efficiency of the invasion of HSC-2 cells was only 1/50 that of KB cells (22). It has recently been shown that KB cells have the capability to produce IL-8 in response to other stimuli, such as neutrophil serine proteinase (34) and IL-1 and gamma interferon (36), and why KB cells did not produce IL-8 after the challenge with A. actinomycetemcomitans Y4 is unknown.

It has been shown that membrane-bound CD14 and sCD14 can bind to gram-negative bacteria (15) and that CD14-expressing monocytes and monocytic cells phagocytose gram-negative bacteria, such as killed Escherichia coli (9, 28) and live A. actinomycetemcomitans (24), by a CD14-dependent mechanism. The CD14-dependent phagocytosis of monocytes was also sustained by the presence of LBP (9, 28). In contrast to serum, saliva does not contain LBP (36). The invasion of oral epithelial cells by A. actinomycetemcomitans is an active process (29) and was inhibited by treatment with cytochalasin B (Fig. 4), indicating that invasion is dependent on actin, as described previously (5). Therefore, the present study suggests that saliva CD14 bound to A. actinomycetemcomitans and promoted the actin-dependent active process by which A. actinomycetemcomitans invades nonphagocytic oral epithelial cells independently of LBP in the oral cavity. It is also possible that LBP from gingival crevicular fluid may facilitate the process of invasion.

The viability of HSC-2 cells after challenge with A. actinomycetemcomitans Y4 and subsequent culture for 24 h was more than 90%, irrespective of the presence of less than 50% saliva, as assessed with the trypan blue exclusion test. A previous report showed that IL-8 mRNA expression is induced by A. actinomycetemcomitans infection (13). These observations indicate that A. actinomycetemcomitans in oral epithelial cells induces signaling pathways in the host cell to produce IL-8, although the underlying mechanism is unclear. It is reported that addition of chloramphenicol, an inhibitor specific for prokaryotic protein synthesis, decreased invasion by A. actinomycetemcomitans of KB cells without affecting the bacterial viability, indicating that bacterial factors are involved in the invasion (29). In support of this, heat-killed A. actinomycetemcomitans showed only marginal IL-8-inducing activity and whole and parotid saliva had no influence on the IL-8 production (data not shown). The results exclude the possibility that LPS of A. actinomycetemcomitans is preventing apoptosis through induction of nuclear factor-B-mediated antiapoptotic factors, resulting in increase in the IL-8 production. Whole and parotid saliva at 100% concentrations had cytotoxic effects on HSC-2 cells, suggesting that saliva contains a cytotoxic or antibacterial component(s) to which cells cultured in in vitro systems are susceptible.

The present study showed that saliva exhibited activity against a low concentration (i.e., 10⁷ CFU/ml) of A. actinomycetemcomitans Y4, but the activity was reduced with higher concentrations (i.e., 10⁸ and 10⁹ CFU/ml) of the bacterium (Fig. 3). There is no report that either membrane-bound CD14 or sCD14 exerts antimicrobial activity, and rCD14 showed no activity against A. actinomycetemcomitans Y4 at 10⁷ CFU/ml (Fig. 3B). Therefore, the observation suggests that the activity of saliva against A. actinomycetemcomitans Y4 indicated by the results shown in Fig. 3A is due to the concerted action of a number of antimicrobial agents other than CD14 in saliva, as suggested previously (14, 33), and that saliva exhibits antibacterial activity in the normal gingival environment when the numbers of periodontopathic bacteria such as A. actinomycetemcomitans are limited.

Saliva CD14 promoted the bacterial invasion of oral epithelial cells and consequently augmented the production of IL-8 by these cells. IL-8 is a major chemokine responsible for the activation of neutrophils and migration of neutrophils and T cells to inflammatory sites (2). Although A. actinomycetemcomitans invasion of oral epithelial cells is considered an important event in the establishment of periodontal disease (22), the present study suggests that when bacteria propagate in the periodontal pocket, saliva CD14 promotes the invasion and induces production of IL-8 by oral epithelial cells to recruit neutrophils and T cells and activate neutrophils for the initiation and establishment of an innate immune response to the bacteria at the site of infection. Furthermore, A. actinomycetemcomitans at extremely high concentrations activates oral epithelial cells independently of saliva components, which may lead to the initiation and development of oral inflammation, i.e., aggressive and chronic periodontitis.

Primary oral epithelial cells as well as HSC-2 and KB cells express TLR2 and TLR4 but not CD14 mRNA (31, 35). However, in contrast to colonic epithelial cells, oral epithelial cells are refractory to many bacterial cell surface components such as LPS, even in the presence of sCD14. Therefore, the CD14-dependent activation of oral epithelial cells by invasive A. actinomycetemcomitans shown in this study reemphasizes the importance of saliva for oral mucosal defense by promoting an innate immune response and for the maintenance of oral health.

 
We thank T. Nishihara for providing the bacterial strain, E. Nemoto (Tohoku University Graduate School of Dentistry) for helpful discussions, and D. Mrozek (Medical English Service, Kyoto, Japan) for reviewing this paper.

This work was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (13671894, 14370576, and 15390551).

 
* Corresponding author. Mailing address: Division of Oral Immunology, Department of Oral Biology, Tohoku University Graduate School of Dentistry, 4-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan. Phone: 81-22-717-8320. Fax: 81-22-717-8322. E-mail: sugawars{at}mail.cc.tohoku.ac.jp.

Editor: J. T. Barbieri

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Infection and Immunity, October 2003, p. 5598-5604, Vol. 71, No. 10
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.10.5598-5604.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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