CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, Mar. 2003, p. 286–292 Vol. 10, No. 21071-412X/03/$08.00�0 DOI: 10.1128/CDLI.10.2.286–292.2003Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Constitutive Expression of a Bacterial Pattern Recognition Receptor,CD14, in Human Salivary Glands and Secretion as a

Soluble Form in SalivaAkiko Uehara,1 Shunji Sugawara,1* Kouichi Watanabe,2 Seishi Echigo,3 Mitsunobu Sato,4

Takahiro Yamaguchi,2 and Haruhiko Takada1

Department of Microbiology and Immunology1 and Department of Oral and Maxillofacial Surgery,3 Tohoku University Schoolof Dentistry, Sendai 980-8575, Department of Animal Production Science, Tohoku University Graduate School of

Agricultural Science, Sendai 981-8555,2 and Second Department of Oral and Maxillofacial Surgery,Tokushima University School of Dentistry, Tokushima 770-8503,4 Japan

Received 23 August 2002/Returned for modification 4 December 2002/Accepted 20 December 2002

Saliva contains a number of proteins and glycoproteins that protect oral tissues, but little is known aboutthe role of human saliva in innate immunity. Here we showed that human major salivary gland cells consti-tutively expressed a bacterial pattern recognition receptor, CD14, by immunohistochemistry. Human salivarygland cells in culture express CD14 mRNA and a 55-kDa CD14 protein in, but not on the cells, and secrete asoluble form with the same molecular mass. Human whole saliva contains a 55-kDa CD14, and the concen-tration of parotid saliva was 10-fold higher than whole saliva, which is comparable to that of serum CD14.Levels of CD14 in unstimulated whole and parotid saliva were unchanged before and after a meal and betweenunstimulated and stimulated saliva, indicating that saliva CD14 is constitutively secreted into the oral cavity.In contrast, lipopolysaccharide (LPS)-binding protein was below the detectable level. The saliva CD14 isfunctionally active in that it mediated the activation of CD14-lacking intestinal epithelial cells by LPS in aToll-like receptor 4-dependent manner. These results suggested that saliva CD14 is important for the main-tenance of oral health and possibly intestinal homeostasis.

Saliva, a complex mix of fluids from major (parotid, subman-dibular, and sublingual) and minor salivary glands, is a mostvaluable oral fluid that is critical to the preservation and main-tenance of oral health. In addition to containing about 99%water, saliva contributes to (i) the lubrication and protection oforal tissues, acting as a barrier against irritants; (ii) bufferingand clearance; (iii) the maintenance of tooth integrity; and (iv)taste and digestion (15). Whole saliva also contains a numberof antimicrobial agents, secretory immunoglobulin (IgA), pro-teins (glycoproteins, statherins, agglutinins, histidine-rich pro-teins, and proline-rich proteins), mucins, lactoferrin, enzymes(lysozyme and peroxidase), and antimicrobial peptides (10,27). The concerted action of these agents is thought to providea multifunctional protective network against microorganisms.

CD14 is a 55-kDa glycosylphosphatidylinositol-anchoredglycoprotein that is expressed mainly on the surface of mono-cytes and macrophages (9). CD14 functions as a bacterial pat-tern recognition receptor for many bacterial components in theinnate immune response to bacterial invasion (18, 30). Re-cently, the family of Toll-like receptors (TLRs) were found tobe essential molecules for microbial recognition in innate im-munity; e.g., TLR2 acts as a receptor for peptidoglycan, zymo-san, and lipoproteins and TLR4 acts as a receptor for lipopoly-saccharide (LPS), taxol, and heat shock protein 60 (1). CD14mediates sensitive responses to LPS by facilitating interactionwith TLR4 in association with MD-2 (1). CD14 also exists in

serum (4) and milk (5, 13) as a soluble form (sCD14). It hasbeen reported that sCD14 in serum decreases cellular re-sponses to LPS by transferring cell-bound LPS to serum li-poproteins and lactoferrin (3, 12) and that sCD14 at a lowconcentration mediates the activation of CD14-negative cells,such as endothelial and epithelial cells, by LPS (6, 17). Wehave recently shown that CD14-lacking epithelial cells respondto LPS in an sCD14-dependent manner to produce interleu-kin-8 (IL-8) (29), a major chemokine responsible for the neu-trophil activation and migration of neutrophils to inflammatorysite (2).

The average daily flow of whole saliva amounts to 1 to 1.5liters, and it has been suggested that another important pro-tective factor is a constant flow of saliva from the mouth intothe intestine (28). Therefore, we speculated that a more spe-cific component responsible for systemic innate immunity act-ing against bacteria might be secreted from salivary glands intosaliva. The present study showed that major salivary glandsconstitutively express and secrete a bacterial pattern recogni-tion receptor, sCD14, into saliva and that salivary CD14 me-diated the activation of CD14-lacking intestinal epithelial cellsby LPS in a TLR4-dependent manner, suggesting that salivaryCD14 is important for the maintenance of not only oral healthbut also intestinal homeostasis.

MATERIALS AND METHODS

Reagents. An ultrapurified LPS preparation from Salmonella enterica serovarAbortus-equi (Novo-Pyrexal) (7) was kindly provided by C. Galanos (Max PlanckInstitut fur Immunbiologie, Freiburg, Germany). All other reagents were ob-tained from Sigma-Aldrich (St. Louis, Mo.) unless otherwise indicated.

* Corresponding author. Mailing address: Department of Microbi-ology and Immunology, Tohoku University School of Dentistry, 4-1Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan. Phone: 81-22-717-8306. Fax: 81-22-717-8309. E-mail: sugawars@mail.cc.tohoku.ac.jp.

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Immunohistochemistry. Human normal parotid and submandibular gland tis-sues were obtained with informed consent from adult patients undergoing sur-gery to remove neighboring tumor tissues. Immunohistochemistry was per-formed as described previously (25), using sheep anti-human CD14 polyclonalantibody (Ab) (Genzyme/Techne, Minneapolis, Minn.). The immunoreactivitywas observed with a confocal laser microscope (Bio-Rad Laboratories, Hercules,Calif.). The serous and mucous cells of the sections were histologically confirmedby periodic acid-Schiff staining. The experimental procedures were approved bythe Ethical Review Board of Tohoku University School of Dentistry (Sendai,Japan).

Cells and cell culture. The human parotid gland cell line HSY (31), the humanneoplastic submandibular gland epithelial duct cell line HSG (21), and thehuman salivary acinar cell line AZA3 (19) were prepared by M. Sato as describedpreviously and grown in Dulbecco’s modified Eagle’s medium (Life Technolo-gies, Grand Island, N.Y.) with 10% fetal calf serum (Life Technologies). Thehuman monocytic cell line THP-1 was obtained from the Health Science Re-search Resources Bank (Osaka, Japan). The human oral epithelial cell lineHSC-2 was obtained from the Cell Resource Center for Biomedical Research,Tohoku University (Sendai, Japan). The human colon adenocarcinoma cell lineSW620 (CCL-227) was obtained from the American Type Culture Collection(Manassas, Va.). THP-1, HSC-2, and SW620 cells were cultured in RPMI 1640(Life Technologies) with 10% fetal calf serum. THP-1 cells were treated with1�,25-dihydroxyvitamin D3 (Biomol, Plymouth Meeting, Pa.) for 3 days to inducematuration and CD14 expression on the cell surface (26).

Human peripheral blood mononuclear cells (PBMCs) were isolated fromheparinized peripheral blood of healthy adult donors by Lympholyte-H (Ceder-lane Laboratories Ltd., Hornby, Ontario, Canada) gradient centrifugation at 800� g for 20 min at room temperature (24).

Collection of saliva. Whole saliva was collected from healthy adult donors, age22 to 24 years, into a sterile plastic tube after the collection of parotid saliva.Parotid saliva was collected with the aid of Schaefer cups placed over theStenson’s duct (20). Stimulated saliva was collected by having the donors chew awax piece. The saliva samples were immediately clarified by centrifugation at

14,000 � g for 5 min at 4°C. Clarified saliva samples were collected, aliquoted,and frozen at �70°C until use.

Reverse transcriptase PCR (RT-PCR). Total cellular RNA was prepared fromhuman cells with Isogen (Nippon Gene, Tokyo, Japan) according to the manu-facturer’s instructions. Random-hexamer-primed reverse transcription was per-formed on 2.5 �l of total RNA in a 50-�l reaction volume, and all PCR proce-dures were performed with a 20-�l volume as described previously (29). Theprimers used for PCR had the following sequences: CD14, 5�-CTCAACCTAGAGCCGTTTAT-3� and 5�-CAGGATTGTCAGACAGGTCT-3�; and humanglyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5�-TGAAGGTCGGAGTCAACGGATTTGGT-3� and 5�-CATGTGGGCCATGAGGTCCACCAC-3�.Cycling conditions were as follows: with CD14, 25 cycles at 94°C for 1 min, 65°Cfor 1 min, and 72°C for 1 min for amplifying a 426-bp product; and with GAPDH,35 cycles at 94°C for 1 min, 60°C for 1 min, and 72°C for 1 min for amplifying a983-bp product. Amplified samples were visualized on 2.0% agarose gels stainedwith ethidium bromide and photographed under UV light.

Western blotting. Cell pellets of human salivary gland cells (3 � 105 cellseach), vacuum-dried 24-h culture supernatant of the cells (equivalent to 3 � 105

cells each), and human saliva (10 �l) were separated by sodium dodecyl sulfate–12% polyacrylamide gel electrophoresis under reducing conditions, and theexpression of CD14 was analyzed by Western blotting as described previously(25). Briefly, proteins were transferred to a polyvinylidene difluoride membraneby using a semidry transblot system (ATTO Instruments, Tokyo, Japan). The blotwas blocked for 2 h with 5% (wt/vol) nonfat dry milk and 0.05% Tween 20 inphosphate-buffered saline (Blotto-Tween) and incubated with 2 �g of sheepanti-human CD14 polyclonal Ab per ml in Blotto-Tween overnight at 4°C. Theblot was washed four times with Blotto-Tween and then incubated for 2 h withhorseradish peroxidase-conjugated affinity-purified donkey anti-sheep IgG(Jackson ImmunoResearch Laboratories, West Grove, Pa.) at 1:2,000 in Blotto-Tween. After the blot was washed, CD14 was visualized with diaminobenzidinein the presence of 0.03% CoCl2. The molecular weights of the proteins wereestimated by comparison with the positions of a standard (Bio-Rad Laborato-ries). Parotid saliva and human recombinant CD14 (rCD14) (Biometec GmbH,

FIG. 1. Expression of CD14 protein in human salivary gland cells. Cryosections of human parotid gland (A and B) and submandibular gland(C and D) tissues were stained with the anti-CD14 polyclonal Ab (A and C) or control Ab (B and D) (green). Cell nuclei were counterstained withpropidium iodide (red). s, serous cells; m, mucous cells. Bars, 20 �m.

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Greifswald, Germany) were adjusted to pH 2.5 with 1 M glycine-HCl (pH 2.5)and treated with or without various doses of pepsin for 30 min at 37°C. Thetreated saliva, which was equivalent to 10 �l of untreated saliva, and rCD14 (20ng) were also analyzed by Western blotting.

Flow cytometry. Flow cytometric analyses were performed with a fluorescence-activated cell sorter (FACScan; Becton Dickinson and Co., Franklin Lakes, N.J.)as described previously (23). Briefly, cells were stained with fluorescein isothio-cyanate-conjugated anti-CD14 monoclonal Ab (MAb) MY4 (mouse IgG2b;Beckman Coulter, Miami, Fla.) or fluorescein isothiocyanate-conjugated isotype-matched mouse IgG (Beckman Coulter) at 4°C for 30 min. The use of isotype-matched Ab excludes the possibility of the nonspecific binding of anti-CD14MAb.

Measurement of CD14, LBP, and cytokine. Cell culture supernatants werecollected from confluent human salivary gland and oral epithelial cells in culturefor 24 h. Parotid saliva was incubated with various doses of Streptococcus sali-varius ATCC 25975 for the times indicated at 37°C and centrifuged at 14,000 �g for 5 min at 4°C. Levels of CD14 and LPS-binding protein (LBP) in thesupernatants and human saliva were measured with a human sCD14 enzyme-linked immunosorbent assay (ELISA) kit (BioSource Europe, Fleurus, Belgium)and a human LBP ELISA kit (HyCult Biotechnology, Uden, The Netherlands).CD14-lacking HSC-2 and SW620 cells (104 cells/200 �l) (29) were seeded inculture medium in the wells of 96-well plates (Falcon; Becton Dickinson andCo.). After incubation for 1 day, the cells were stimulated with LPS in thepresence or absence of human saliva at various doses or 50 ng of rCD14 per mlin 200 �l of the medium without serum for 24 h. Cells were pretreated withanti-CD14 MY4, anti-TLR2 TL2.1 (mouse IgG2a; Cascade BioScience, Win-chester, Mass.), anti-TLR4 HTA125 (mouse IgG2a; Medical & Biological Lab-oratories, Nagoya, Japan), or isotype control IgG (10 �g/ml each) for 30 min, andthe MAbs remained present during incubation for 24 h. The amounts of IL-8 inthe supernatants were measured with an OptEIA ELISA set (PharMingen, SanDiego, Calif.). The concentrations of CD14, LBP, and IL-8 were determined by

using the Softmax data analysis program (Molecular Devices Corp., Menlo Park,Calif.).

Data analysis. All experiments in this study were performed at least threetimes to confirm the reproducibility of the results. For most experiments, valuesare represented as means � standard deviations from triplicate assays. Thestatistical significance of differences between the two means was evaluated byone-way analysis of variance, using the Bonferroni or Dunn method, and P valuesof less than 0.05 were considered significant.

RESULTS

Human salivary gland cells express and secrete CD14. Im-munohistochemistry showed that CD14 protein was present inthe acinar and intercalated duct cells of tissues from humanparotid gland, a serous gland (Fig. 1A and B). The CD14 wasmore abundant in the intercellular secretory capillary of acinarcells and the duct region than in acinar and intercalated ductcells. In the section of human submandibular gland, a mixedgland of serous and mucous cells, CD14 was expressed in onlyserous cells and not in mucous cells (Fig. 1C and D). Theseresults indicate that CD14 is secreted from serous acinar andintercalated duct cells into the salivary duct. We next examinedthe expression of CD14 by using human parotid gland inter-calated duct HSY cells, human submandibular gland interca-lated duct HSG cells, and acinar AZA3 cells in culture. Weused PBMCs, which include CD14� monocytes, and CD14-expressing THP-1 cells as positive controls and human oralepithelial HSC-2 cells as a negative control. The results showedthat HSY, HSG, and AZA3 cells, but not HSC-2 cells, consti-tutively expressed CD14 mRNA as detected by RT-PCR anal-ysis (Fig. 2A) and CD14 protein of a 55-kDa form as deter-mined by immunoblot analysis (Fig. 2B). Furthermore, CD14was detected in 24-h culture supernatants of the salivary glandcells with the same molecular mass as that in the cell lysates(Fig. 2B) at levels of 100 to 200 ng/ml as assessed by ELISA(Fig. 2C). In contrast, no expression of CD14 on the surface ofsalivary gland cells in culture could be detected by flow cytom-etry (Fig. 2D). These results indicate that CD14 is secretedconstitutively from salivary gland cells into saliva.

Detection of CD14 in human saliva. We then examined thepresence of CD14 in human saliva. In whole saliva, the 55-kDaform of CD14, with the same molecular mass as in humanmonocytes, was detected by immunoblot analysis (Fig. 3A). Torule out the possibility that CD14 in whole saliva is fromserum-derived gingival crevicular fluid, parotid saliva was col-lected and analyzed. The results showed that 55-kDa CD14was more abundant in parotid saliva than whole saliva (Fig.3A). The levels of CD14 in unstimulated whole and parotidsaliva as assessed by ELISA were 0.19 � 0.01 �g/ml (n � 8)and 1.44 � 0.97 �g/ml (n � 6), respectively (Fig. 3B). Further-more, to rule out the possibility that CD14 in saliva is derivedfrom serum, LBP, which is abundant in serum, was examined.In contrast to the case for human serum, the amount of LBP(the serum protein that accelerates the binding of LPS tosCD14 [8]) in both saliva preparations was below detectablelevels (20 ng/ml). Parotid saliva contains 10 times moreCD14 than whole saliva, indicating that the parotid gland is amajor source of the CD14 in whole saliva. The amounts ofCD14 in unstimulated whole and parotid saliva were un-changed before and after a meal and between unstimulated

FIG. 2. Expression and secretion of CD14 in human salivary glandcells in culture. (A) HSY, HSG, AZA3, and oral epithelial HSC-2 cellswere collected from confluent cultures. Total RNA was extracted fromthese cells, and the mRNA expression of CD14 and GAPDH wasanalyzed by RT-PCR. (B) Whole-cell lysates and 24-h culture super-natants of PBMCs and HSY, HSG, AZA3, and HSC-2 cells weresubjected to Western blotting with an anti-CD14 polyclonal Ab.(C) The amounts of sCD14 in the 24-h culture supernatants of the cellswere measured by ELISA. (D) HSY, HSG, and AZA3 cells werestained with anti-CD14 MAb or isotype control IgG and then analyzedby flow cytometry. Vitamin D3-treated THP-1 cells were used as apositive control.

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and stimulated saliva (Fig. 3C), indicating that saliva CD14 isconstitutively secreted into the oral cavity.

Biological functions of saliva CD14. Since parotid saliva is amajor source of whole saliva (10), there is a quantitative dis-crepancy in Fig. 3, which shows that whole saliva contains 10times less CD14 than parotid saliva. To examine whether salivaCD14 can bind to oral bacteria, parotid saliva was incubatedwith S. salivarius, a major bacterium in saliva. The resultsshowed that saliva CD14 was significantly absorbed by S. sali-varius in a time- and dose-dependent manner (Fig. 4A). Wenext analyzed whether saliva CD14 confers activation of CD14-lacking oral epithelial HSC-2 cells and intestinal epithelialSW620 cells by bacterial components, as is the case for serum-derived sCD14 (20). LPS (100 ng/ml) alone did not activateHSC-2 and SW620 cells, whereas saliva CD14 mediated theactivation of SW620 but not HSC-2 cells by LPS in a salivadose-dependent manner to produce IL-8 (Fig. 4B). The salivaCD14-mediated activation of SW620 by LPS was significantlyinhibited by anti-CD14 MY4, and anti-TLR4 HTA125 but notby anti-TLR2 TL2.1, which was consistent with the resultsobtained with 50 ng of rCD14 per ml as a control (Fig. 4C).These findings clearly indicate that saliva CD14 is functionallyactive. Furthermore, saliva CD14 was partly degraded by pep-

sin treatment at pH 2.5, but, in contrast to rCD14, it wassubstantially resistant to the treatment with even 20 mg ofpepsin per ml (Fig. 4D), suggesting that saliva CD14 exhibitsbiological activity not only in the oral cavity but also in theintestine.

DISCUSSION

The present study showed for the first time that a bacterialpattern recognition receptor, CD14, is constitutively expressedin serous acinar and intercalated duct cells of the human majorsalivary gland and secreted directly into saliva, which was con-firmed by using human salivary gland cells in culture, and thatsaliva CD14 mediates the activation of CD14-lacking intestinalepithelial cells by LPS.

CD14 protein was present in the acinar and intercalatedduct cells of tissues from human parotid gland, a serous gland(Fig. 1A). Human submandibular and sublingual glands aremixed glands of serous and mucous cells (10, 15, 28), andCD14 protein was present only in serous cells and not inmucous cells of tissues from the submandibular gland as de-termined by immunohistochemistry (Fig. 1C). The concentra-tion of CD14 in parotid saliva was 1.44 � 0.97 �g/ml (Fig. 3B),

FIG. 3. Detection of sCD14 in whole and parotid saliva. (A) Whole saliva and parotid saliva (10 �l each) from three donors (donors 1 to 3)were subjected to Western blotting with anti-CD14 polyclonal Ab. A preparation of PBMCs was loaded as a control. The results are representativeof those from four different experiments with similar results. (B) The amounts of sCD14 (closed symbols) and LBP (open symbols) in whole andparotid saliva from different donors were measured by ELISA. (C) Unstimulated whole and parotid saliva from different donors before and aftera meal and stimulated whole and parotid saliva from the same donors were collected. The amounts of sCD14 (closed symbols) and LBP (opensymbols) in the saliva were measured by ELISA.

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which was comparable to that in normal serum (1 to 2 �g/ml)(12) and 10-fold the amount in whole saliva (Fig. 3B). Theseresults indicate that the major source of saliva CD14 was se-rous acinar and intercalated duct cells in the parotid gland. Incontrast to the case for serum, the levels of LBP in whole andparotid saliva were below the detectable limit (Fig. 2B and C).The results exclude the possibility that saliva CD14 originatedfrom serum-derived gingival crevicular fluids. Stimulated salivacontributes as much as 80 to 90% of the average daily salivaryproduction, and parotid saliva contributes more than 50% ofstimulated saliva (10). Taking into account this information,the amount of CD14 in whole saliva should be more than thelevels shown in Fig. 3. One possible explanation for the dis-crepancy is that saliva CD14 is absorbed by bacteria in oralflora, since saliva CD14 was absorbed by a major bacterium insaliva, S. salivarius (Fig. 4A). The molecular mass of CD14expressed in salivary cell lines and released from the cells wasidentical to that of glycosylphosphatidylinositol-anchored

CD14 on monocytes (Fig. 2B), which is also the case for CD14from parotid saliva as well as whole saliva (Fig. 3A). Thesefindings indicated that the protein portion of saliva CD14 wasnot truncated and that saliva CD14 is synthesized as a 55-kDaform in salivary glands and secreted as a protein of the samesize as in the case of human hepatocytes (22), the major sourceof sCD14 in serum (16).

It has been reported that sCD14 is abundant in serum andthat sCD14 in serum decreases monocyte responses to LPS bytransferring cell-bound LPS to plasma lipoproteins by shiftingthe equilibrium between LPS-membrane CD14 and LPS-sCD14 toward sCD14 (12). It also has been reported thatsCD14 interacts with lactoferrin and LPS in serum and inhibitsendothelial cell activation induced by the CD14-LPS complex(3). Lactoferrin is abundant in saliva as well as in plasma as anonimmune antimicrobial factor (10, 15, 28). However, trans-ferrin, a lactoferrin homologue, is found in plasma but not insaliva. Therefore, it is possible that saliva CD14 interacts with

FIG. 4. Biological functions of saliva CD14. (A) Parotid saliva was incubated with given doses of S. salivarius for the indicated times at 37°Cand centrifuged. The amounts of CD14 in the saliva were then measured by ELISA. (B) HSC-2 and SW620 cells were incubated with the indicateddoses of parotid saliva in the presence or absence of LPS (100 ng/ml) for 24 h. (C) SW620 cells were stimulated with LPS (100 ng/ml) in thepresence of either parotid saliva (2%) or rCD14 (50 ng/ml) for 24 h. Cells were pretreated with anti-CD14 MY4, anti-TLR2 TL2.1, anti-TLR4HTA125, or isotype control IgG (10 �g/ml each) for 30 min, and the MAbs remained present during the incubation for 24 h. The amounts of IL-8in the supernatants were measured by ELISA. ��, P 0.01 compared with the respective controls (parotid saliva without S. salivarius [A], parotidsaliva without LPS [B], or LPS with saliva or rCD14 [C]). Error bars indicate standard deviations. (D) rCD14 and parotid saliva were treated withor without the indicated doses of pepsin for 30 min at 37°C and subjected to Western blotting with anti-CD14 polyclonal Ab.

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bacterial components, including LPS and lactoferrin, and pre-vents the oral mucosa and salivary glands themselves fromexacerbating innate immune responses by clearing bacterialcomponents. It also has been reported that sCD14 mediatesthe aggregation of LPS and that the aggregates undergo inter-nalization by phagocytes, which do not elicit cellular responsesto the LPS (11). This observation suggests that saliva CD14participates in the aggregation of LPS and possibly many otherbacterial components, since CD14 functions as a bacterial pat-tern recognition receptor, consequently contributing to theclearance of bacteria in the oral cavity to maintain oral health.It is speculated that more than 400 bacterial species reside inoral cavity and that 1011 bacteria, corresponding to 1 g (wetweight), exit from the oral cavity in saliva per day (14). Theabsorption of saliva CD14 by S. salivarius, as shown in Fig. 4A,may also contribute to the clearance. Furthermore, in contrastto intestinal epithelial cells, oral epithelial cells were unrespon-sive to LPS and many other bacterial components even in thepresence of saliva CD14 and rCD14 (Fig. 4B and C) (29),which may be necessary to avoid excessive innate immuneresponses to oral bacteria.

Another protective factor is the constant flow of saliva fromthe mouth into the gut, not only because a steady supplyguarantees the continuous presence of both nonimmune andimmune factors in the mouth but also because it efficientlyremoves exogenous and endogenous microorganisms and theirproducts into the gut (27). The average daily flow of wholesaliva in healthy individuals amounts to 1 to 1.5 liters. It is ofinterest that saliva CD14 is constitutively secreted irrespectiveof food intake or stimulation (Fig. 3C). According to our es-timates, the amount of saliva CD14 secreted daily into the oralcavity, where it flows into the gut, is on the order of milligrams.Saliva CD14 was relatively resistant to pepsin (Fig. 4D), prob-ably due to the presence of saliva proteins. It also has beenreported that a low concentration of sCD14 or diluted serum inturn mediated the activation of CD14-negative endothelial andepithelial cells by LPS (6, 17), and the present study showedthat saliva CD14 mediated the responsiveness of CD14-lackingintestinal epithelial cells to LPS (Fig. 4B and C). Therefore,another role of saliva CD14 may be the maintenance of ho-meostasis in intestinal mucosa. The possibility is supported bythe recent report of the existence of milk CD14 (5, 13); thereport suggested that milk CD14 is involved in controllinghomeostasis in the neonatal intestine. Concentrations of sali-vary antimicrobial agents, such as lysozyme and peroxidase,and secretory IgA already reached adult levels by age 6 monthswhen the primary teeth emerged, and lactoferrin and myelo-peroxidase concentrations were lower in children than inadults (28). These findings provide for the possibility that salivaCD14 takes over the role of milk CD14 after breastfeeding,although the levels of CD14 in saliva at different ages, fromnewborn babies to the elderly, remain to be elucidated.

Finally, the present study may suggest that CD14 in solutionplays an important role in the regulation of reactions to LPSand other bacterial components not only within the blood-stream but also within the oral cavity and may furtherstrengthen the importance of saliva in the preservation andmaintenance of oral health and possibly intestinal homeostasis.

ACKNOWLEDGMENTS

We thank C. Galanos for providing an LPS preparation and Y. Yura(Osaka University, Osaka, Japan) and M. Okamoto (Tokushima Uni-versity, Tokushima, Japan) for supplying HSY, HSG, and AZA3 cells.

This work was supported in part by Grants-in-Aid for ScientificResearch from the Japan Society for the Promotion of Science(12470380, 13671894, and 14370576).

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CLINICAL AND VACCINE IMMUNOLOGY, Apr. 2010, p. 698 Vol. 17, No. 41556-6811/10/$12.00 doi:10.1128/CVI.00092-10Copyright © 2010, American Society for Microbiology. All Rights Reserved.

RETRACTION

Constitutive Expression of a Bacterial Pattern Recognition Receptor, CD14, inHuman Salivary Glands and Secretion as a Soluble Form in SalivaAkiko Uehara, Shunji Sugawara, Kouichi Watanabe, Seishi Echigo, Mitsunobu Sato,

Takahiro Yamaguchi, and Haruhiko TakadaDepartment of Microbiology and Immunology and Department of Oral and Maxillofacial Surgery, Tohoku University School of

Dentistry, Sendai 980-8575, Department of Animal Production Science, Tohoku University Graduate School ofAgricultural Science, Sendai 981-8555, and Second Department of Oral and Maxillofacial Surgery,

Tokushima University School of Dentistry, Tokushima 770-8503, Japan

Clinical and Diagnostic Laboratory Immunology, volume 10, no. 2, pages 286–292, 2003. The publisher hereby retracts this article.An investigation by Tohoku University has concluded that images in the paper were misappropriated.

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