Oral Med Pathol 11 (2006) 1

Review in Saliva and Salivary Glands: Their Roles in the Oral Defense System

Masahiko Mori1, Hiroshi Takeuchi2, Masaru Sato2 and Shinichiro Sumitomo1 1 Department of Oral and Maxillofacial Surgery, 2Department of Oral Pathology, Asahi University School of Dentistry, Gifu, Japan

Mori M, Takeuchi H, Sato M and Sumitomo S. Antimicrobial peptides in saliva and salivary glands: their roles in the oral defense system. Oral Med Pathol 2006; 11: 1-17, ISSN 1342-0984

The majority of inflammatory diseases in the oral cavity arise from infections caused by several oral microorganisms inhabiting the biofilms formed on the surfaces of teeth, prosthetic devices, and oral mucosa. Human whole saliva is a mixture of secreted saliva from major and minor salivary glands. In addition, it also contains components derived from crevicular fluid. A number of families of peptides, such as cystatins, , statherins, lipocalins (VEG ), chromogranins, calprotectins and , are found in whole saliva. In recent years, much attention has been focused on these peptides because they show antimicrobial activity against oral pathogens. These naturally occurring antimicrobial peptides are anticipated to be potent therapeutic agents for oral infectious diseases because the acquision of microbial resitance to antibiotics is one of the most serious problems for antibiotic therapy. The present paper reviews recent findings of studies on antimicrobial peptides found in saliva and salivary glands, with special reference to their nature and function in maintaining oral health. We further discuss the methodology in basic research on antimicrobial peptides as well as the possibility of their clinical use in oral health care science.

Key words: antimicrobial peptide, oral health, defense system, saliva, salivary glands Correspondence: Masahiko Mori, Department of Oral and Maxillofacial Surgery 1851-Hozumi, Mizuho, Gifu 501-0296, Japan Phone and Fax: + 81-58-329-1472, E-mail: [email protected]

Introduction for microorganisms to adhere firmly onto the surfaces of The mucosal surfaces of the oral cavity are covered teeth and epithelial cells. Biofilms consist not only of mi- with sialoproteins, including immunoglobulins, biologi- croorganisms but also of complex structures of salivary cally active peptides, cytokines and growth factors mucins, food related materials, and exfoliated (sialomucinous coating). Sialomucins are composed keratinocytes. Biofilms also contain leukocytes and mono- mainly of alpha-glycosylated glycoproteins synthesized cytes from gingival crevicular fluid and/or peptides de- from acinar cells in the major and minor salivary glands. rived from these cells. Several pathogenic bacteria in the In addition to the well-known sialoproteins (lysozyme, biofilm produce bacterial toxins and cytokines, and host lactoferrin and peroxidase), human whole saliva contains tissues respond to the stimuli with inflammation. Thus, many other peptides with bactericidal and fungicidal ac- infections in the oral cavity are considered to be protec- tivities (antimicrobial peptides). Antimicrobial peptides tive responses not only for local tissue destructions but were originally discovered in insects with cell-free also for systemic diseases. A new method combining mo- hemolymph, which corresponds to blood in mammals. lecular and immunological techniques has been developed Recently, it has been reported that antimicrobial peptides to investigate biofilm-specific surface protein expression are involved in the innate immunity of mammals and that by Streptococcus sanguis (1), and the results have shown they function in the recognition of pathogens by signal- that surface expression of putative fibronectin and col- ing pathway and effector mechanisms. lagen adhesions is upregulated by biofilm cells. Biofilms Dental biofilms are formed under an oral environ- are resistant to antimicrobial agents. In addition, phago- ment, in which a vast variety of microorganisms become cytic cells cannot phagocytose or kill adhesive bacteria in established and proliferate. Biofilms also make it possible biofilms. Cardiac coronary stenotic artery diseases, as well 2 Mori et al. Antimicrobial peptides in saliva as atherosclerosis caused by periodontal disease-associ- Cystatins are induced in the submandibular (SM) ated bacteria, pose significant health problems. The de- gland of rats treated with β -adrenergic agonists velopment of biofilms containing periodontal disease- (isoproteronol) (9, 10), and high levels of cystatin S mRNA associated bacteria can be prevented by salivary histatins also persist in the rat SM gland (11). Moreover, 5000-fold and cystatins, and thus they may contribute to preven- increase in the cystatin levels were found in isoproter- tion of vascular diseases (2). In order to maintain good enol-treated rats (9), and papain administrated to rats oral health or oral-health-related quality of life (3), new induced cystatins in their salivary glands (12). approaches against oral infectious diseases are urgently Immunohistochemical identification of cystatin has needed. The application of naturally-occurring antimicro- been investigated, and cystatin C and S in the rat SM bial peptides could be a potent program in the prophy- gland showed intense staining in acinar cells but not in laxis and treatment of such oral infectious diseases as ductal cells. Furthermore, isopreterenol-treated rats dental caries, periodontal diseases, peri-implantitis, oral showed high cystatin activity in acinar cells (13). These ulceration, candidiasis, leukoplakia and denture stoma- findings suggest that salivary cystatin could be secreted titis. Almost all such diseases show, at least in some forms, from acinar cells. Rat cystatins in the SM gland are in- features of bacterial or fungal infections. duced by β-adrenergic agonist and correspond to human Although a monograph has been published which cystatin S (14), i.e. LM protein (large mobile protein), a addresses the role of antimicrobial agents in periodontal protein with MW 13,500 that is expressed in both granu- prevention, therapy and maintenance (4), we have cho- lar convoluted tubules (GCT) and acinar cells in normal sen to focus on the key roles of antimicrobial peptides rat SM glands. Upon testosterone administration to rats, from 3 sources: salivary glands, oral epithelial cells, and hypertrophic GCT cells showed marked staining for LM leucocytes or monocytes. We discuss the possibility of the protein, and enlarged acinar cells treated with prolonged utilization of antimicrobial peptides as new preventive isoproterenol were stained most intensely for LM pro- and/or therapeutic agents against oral infectious diseases tein (15, 16). Cystatins (S, SA and SN) found in whole caused by commensal pathogens. This review article also saliva are secreted from the SM-sublingual (SL) glands deals with the immunohistochemical localization of anti- and, to a lesser extent, from the parotid. Henskens et al. microbial peptides from various sources in the oral cav- (17) reported that total cystatin activity in saliva is 5 times ity. higher in SM saliva than in parotid saliva, and cystatins S and C are found in only SM and SL saliva. Cystatins Cystatin levels in inflamed human gingiva range Cystatins are inhibitors of cysteine proteinases, and from 0.21 to 3.82 μg/g tissue as revealed by enzyme- are widly distributed in human tissues and body fluids, linked immunosorbent assay, and cystatin concentrations including saliva. Cystatins are classified into 3 subfami- are decreased in samples with increased pocket depths lies: family I cystatins are represented by cystatin A (stefin (18). Aguirre et al. (19) reported that cystatins in SM-SL A) and cystatin B (stefin B), both of which are non- saliva show a mean value of 129.7 ± 100.9 μg/ml for glycosylated that lack disulfide bonds and con- subjects without periodontal diseases and 92.0 ± 58.1 tain about 100 amino acid residues. Family II cystatins μ g/ml for subjects with periodontal diseases, and that consist of C, D, S, SA, M and F. They are proteins of a there is no statistically- significant difference between single polypeptide chain with disulfide bonds and are these two groups in terms of cystatin level. In contrast, composed of 115-120 amino acids. Family III cystatins Henskens et al. (20) showed that cystatin concentrations are composed of L- and H-kininogens containing in whole saliva of periodontitis patients are significantly glycosylated cytoplasmic proteins with bradykinin moi- higher than those in healthy subjects. It has further been ety. Family III cystatins of high molecular weight are reported that an increase in cystatin levels in whole sa- kininogens and are subdivided into high molecular weight, liva of periodontal disease patients is in part due to in- low molecular weight and tissue kininogens. Cystatins creased salivary gland secretions of both isoform cystatin are distributed in several mammalian species including S and basic cystatin C (17). Cystatins and their synthetic humans, and are also derived from other sources, such as derivatives are capable of blocking bacterial and viral rep- plants, protozoa, bacteria and viruses. Human cystatins lication and inhibiting the chemotactic response, and include cystatins A, B, C, D, S, SA, SN, L-kininogen, and could modulate immune responses in mucosal tissue. H-kininogen in order of their molecular weight (5). These Thus, human salivary cystatin is considered to play a role cystatins are mainly distributed in secretory fluid and in the protection of periodontal tissues. They are also able are highly expressed in human saliva by acidic (S, SA), to modulate the formation of acquired enamel pellicles neutral (SN, D) and basic (C) cyatatins. Family and mineralization processes at the saliva-enamel sur- IIIcystatins (L- and H-kininogens) are intravascular pro- face interface by inhibiting the transformation of teins (6). RNAs for cystatins (7) and full-size forms of dicalcium phosphate dehydrate. cystatins (SN, SA, and S) have been described (8). In order to produce a full-length unaltered human Oral Med Pathol 11 (2006) 3 salivary cystatin SN and its variants, a baculovirus ex- between cystatin C and inactivated papain or actinidin pression system has been used, and groups of predicted decreases with the increasing size of the inactivating proteinase-binding regions (N-terminus, and β -hairpin group in a manner similar to that reported in chicken- loops I and II ) were constructed. The resulting purified inhibitor studies. The mechanism underlying the reac- proteins were then examined for papain and cathepsin-C tions of cystatin C and chicken cystatin is very similar if inhibition. The salivary cystatin SN variants were shown not identical (27). Procathepsin B is activated into ma- to exhibit different effects toward different cysteine pro- ture enzymes within an acidic compartment either by teinases (21). Increased secretions of cystatins C, S and active cathepsin B itself or by cathepsin D. Purified cathe- SN in patients with inflamed gingiva and periodontitis psin D can activate latent cathepsin B isolated from ma- would contribute to maintaining oral health. It is reported lignant ascetic fluid. Cathepsin D can degrade cystatin that cystatin C is a potent inhibitor of mineral mobiliza- C, both of which are secreted in biological fluid. A study tion (Ca 2 ＋). Bone resorption stimulated by parathyroid of 10 human colon carcinoma cell lines has revealed that hormone is inhibited by cystatin C (30 ng/ml), Thus the cell lines secrete cathepsin B and cystatin C. More- cystatin C is a potent inhibitor of bone resorption (22). over, cystatin C accumulates in cell-conditioned media Human cystatin A is localized in the upper epider- together with latent cathepsin B, which is a proenzyme mal layer. Although cystatin has a defence function rather than an enzyme inhibitor complex (28). Human against exogenous pathogens, candidal aspartic protein- bronchial epithelia synthesizes and secretes cystatin C ase (CAP), a putative virulence factor, is not affected by and procathepsin B. The cleavage of both cystatin C and epidermal cystatin A. Ninety percent of cystatin A is lost procathepsin B by neutrophil elastase is essential for the within 12 h after incubation with CAP. It is considered generation of cathepsin B activity (29). Cathepsin B, which that CAP cleaves and inactivates human epidermal cys- can hydrolyze proteins of the extracellular matrix, is teine protenase inhibitor, cystatin A (23). CAP has been stored in the lysosome of macrophages, fibroblasts and reported to degrade extracellular proteins, collagen, and hepatocytes, and participates in inflammation. keratin, and thus candidal infection is found in leuko- Recently, it has been shown that cystatin M, a cathe- plakia and other oral mucosal lesions. Phosphorylated psin B inhibitor, is expressed 40-fold higher in the meta- cystatin α is expressed in the epidermis and is incorpo- static squamous cell carcinoma cell line than in the pri- rated in keratohyalin granules. Furthermore, cystatin α mary tumor cell line. This notable result suggests that is conjugated with glutamine-rich protein as filaggrin, elevated cystatin M activity aids metastasis by blocking suggesting that it has an inhibitory effect against bacte- intrinsic cathepsin B activity and rescuing tumor cells rial and viral cysteine proteinases. Cystatins in the corni- from TNF-induced apoptosis (30). fied envelope are considered to have a barrier function against bacterial infection (as seen, e.g., in the cystatin A Histatins decreases in atopic dermatitis) as well as against fungal Histidine-rich polypeptides have been isolated and and viral infections (e.g., in the replication of Herpes sim- purified from human parotid saliva and they contained plex blocked by cystatin C) (24). 38 amino acid residues. The distribution of hydrophilic Cystatin D contains 142 amino acid residues, its and hydrophobic residues in the polypeptide chain of sequences include a putative signal peptide, and it shows shows no structural polarity (31). Histatins 1, 3 51-55% identical residues with the sequences of cystatins and 5 consist of 38, 32 and 24 amino acid residues, re- C, S, SN and SA. Northern blot analysis of cystatin D has spectively, whereas histatins 2, 4, 6 and 7-12 are polypep- shown that its is expressed in the parotid gland but tides (31). Histatin contains a phosphorylated serine at not in the prostate, epidermis, testis, ovary, placenta, gas- residue 2, and histatins 3 and 5 lack inorganic phosphate tric corpus or small intestine. Cystatin D is clearly present (32). Among the many histatin types, histatins 1, 3 and 5 in the human parotid gland (25). Immunoenzymatic are the major members of the histidine-rich protein fam- analyses of cystatin D revealed that it is present in hu- ily in human salivary secretions. Salivary proteins include man saliva and tears at concentrations of 3.8 and 0.5 mg/ acidic and glycosylated proline-rich proteins, cystatins and l, respectively, whereas it is not found in blood plasma, statherin. They show a high affinity for hydroxyapatite, milk or cerebrospinal fluid (25). In a study of the inhibi- a prototype in the mineral phase of teeth and bones, and tory specificity of human cystatin C and D, it has been are related to pellicle precursor proteins. Histatins are reported that both the N-terminal and framework parts low-molecular-weight, unique pellicle precursor proteins of the molecules significantly contribute to differences in that exhibit both antibacterial and antifungal/ their activities and that the N-terminal segment of anticandidal activities. (33, 34). Oral candidiasis is asso- cystatin C increases its inhibitory activity against cathe- ciated with increased levels of salivary histatin (35). It psin S and L (26). has been reported that histatin 5 inhibits the energized Cystatin C is present in body fluids and is the most mitochondria of Candida albicans (36). Histatin 5 re- important inhibitor of cysteine proteinases. The affinity strains the induction of inflammatory cytokines in gingi- 4 Mori et al. Antimicrobial peptides in saliva val fibroblasts by Porphyromonas gingivalis (37). Further- suggested that the precursor cells of salivary gland tu- more, salivary histatins inhibit both bacterial and host mors secrete histatin protein (49). enzymes in periodontal lesions (38). The absorpotion and Statherin isolation of histatin 5 from hydroxtapatite has been con- Statherin is a 43 amino-acid residue phosphopro- firmed by hydroxyapatite chromatography (39). The in- tein of unusual composition which is secreted from the teraction of histatin 5 with hydroxyapatite causes a re- major salivary gland in humans. The name Statherin, duction in the anticandidal activity and killing efficiency derived from the Greek “statheropio” meaning “to stabi- of histatins (40). Synthetic histatin analogues or recom- lize”, is the only salivary protein that inhibits both pri- binant histatin 1 have been chemically synthesized to mary and secondary calcium phosphate precipitations. produce the C-terminal fungicidal domain of histatin 5 The supersaturation of saliva with calcium makes it pos- (dh-5) (41). Although the recombinant histatin shows sible to stabilize and protect enamel surfaces and candicidal activity comparable to that of native histatin remineralize enamel already demineralized. Enamel pel- 1, it exhibits substantially weaker binding to hydroxya- licle is a protein film consisting of salivary proteins se- patite (42). A synthetic histatin analog causes a signifi- lectively absorbed onto the enamel surface, on which oral cant reduction in the viable counts of bacteria in the microorganisms establish themselves in the initial stage biofilm formed on tooth surfaces (43). Human salivary of plaque formation. Enamel pellicle also acts as a neces- high-molecular mucin MG1 exists as a heterotypic com- sary medium for minerals during recalcification . Li et al. plex with small proteins. The heterotypic complex is (50) have shown that statherin represents the integral thoght to act as a repository for precursors of enamel pel- part of proteins that constitute the pellicle structure and licles (44). Salivary histatins are usually present in may play a key role in its formation. The amino acid se- healthy adults at concentrations of 50-425 μg/ml (45), quence of statherin has been determined, and the NH2- and histatin 5 inhibits candidal pathogenic activity by terminal residues of statherin are all anionic. In addition 90-100% at physiological concentration (34, 36). It has to human saliva, saliva from the monkey, rat, and ham- also been shown that oral candidiasis is associated with ster also inhibit calcium precipitation (51). It is interest- increased levels of salivary histatin (35). Histatin 5 also ing to note that, throughout the animal kingdom, the sa- inhibits bacterial proteases implicated in periodontal dis- liva secreted from salivary glands contains inhibitors of ease at physiological concentration (38). Since histatin 5 calcium precipitation, suggesting a process of evolution- binds significantly to salivary metal, it is considered to ary development (51). Salivary proteins include acidic be a matallopeptide (46). It has recently been reported proline-rich proteins, such as statherin, histatins, and that mean histatin (1, 3 and 5) concentrations are almost cystatins, though statherin is the only protein not known 3 times higher in SM-SL saliva than in parotid stimu- to be polymorphic (52). The physiological function of lated saliva. Daily variations of salivary histatin concen- statherin in human salivary glands is to inhibit the pri- trations are also detected using a histatin-zinc precipita- mary precipitation of calcium phosphate salts of calculi tion method (47). Lysozyme, lactoferrin, and lactoperoxi- in the duct, and to control crystal growth on enamel sur- dase show cytotoxic properties against bacteria and fungi, faces by the epitavic repair of caries lesions and the main- while histatin 5 additionally shows antiviral activity. The tenance of integrity of the mineralized tooth matrix (53, findings described above indicate that histatins play a 54). The concentration of statherin (12.8 μM: mean value) key role in the innate host defense mechanism in the oral in stimulated human parotid saliva plays a significant cavity. role in a system which provides a protective and repara- The immunohistochemical localization of histatin tive environment for the enamel surface (55). Secretory protein has been reported using colloidal gold conjugated rates of statherin and histatin are significantly reduced with anti-human histatin (goat polychonal antiserum). by β-adrenolytic agent (metoprolol) (statherins: 11-29%; Histatin immunoreactivity is confined to the secretory histatins: 58-72% versus the control), but not by α-adre- granules in the SM and parotid acinar cells (48). An in nolytic agent (prazosin). Protective salivary functions in situ hybridization study using digoxigenin-labeled DNA the oral environment may be compromised during β- probe and biochemical analysis of histatin S and C in the adrenolitic treatment (56). In the immunoquantification rat SM glands revealed that both histatins have a 48% of statherin, the level was about 20-times higher in pa- similarity at the nucleotide level. They are localized in rotid and SM-SL saliva than in cleared whole saliva su- serous acinar cells, but are differently regulated in the pernatant or pellicle, indicating the rapid degradation of same cells (13). The immuno-histochemical expression of statherin in the oral cavity (50). Recently, Contucci et al. histatin 5 has also been investigated in detail, revealing (57) investigated statherin levels in saliva of patients with that the duct cells of all salivary glands are stained in- precancerous and cancerous lesions of the oral cavity. They tensely and that duct-like tumor cells and modified myo- found a sensible reduction of statherin level in patients epithelial tumor cells or plasmacytoid cells in pleomor- with precancerous and cancerous lesions compared with phic adenoma show positive reactivity to histatin. It is healthy subjects. Thus, statherin may exhibit a protec- Oral Med Pathol 11 (2006) 5 tive effect in the oral cavity in association with its other These findings suggest that ductal segments in the nor- functions, such as maintaining the integrity of teeth, pro- mal salivary gland, as well as those found in von Ebner’s moting selective initial bacterial colonization, and influ- minor gland, are positive for lipocalin immunoreactivity encing the transport of calcium and phosphate ions dur- (79). In rat lingual epithelium, keratin 14 has been dem- ing secretion in salivary grands (58). onstrated immunohistochemically in keratinocytes of the filiform papillae in the expanding interpapillar regions Lipocalins during morphogenesis (80), suggesting that taste-bud for- Lipocalins are involved in the binding and trans- mation may be related to lipocalin binding. The lipocalin port of small lipophilic ligands, including retinol, steroids, is secreted from the von Ebner’s salivary glands, and thus odorants (retinol binding protein: RBP), bilin binding pro- its expression in whole saliva probably originates from tein, and β-lactoglobulin. Blaker et al. (59) first reported the salivary glands. It is hypothesized that VEG protein that the primary sequence of the human von Ebner’s gland in saliva shows specific gustatory effects by binding lipo- (VEG) protein is similar to that of the lipocalin super- philic bitter compounds and transporting them to the family that consists of 20 different proteins present in taste buds, and that it also shows lipid-binding proper- various structures. Immunohistochemical and in situ ties with oil- soluble substances. It is also been consid- hybridization techniques showed VEG protein reactivity ered that tear binds to retinol or vitamin A derivatives in in acinar cells of the VEG in the tongue, suggesting a protecting cover epithelium and OBP II in nasal secre- close physiological relationship to gestation in the taste tory materials may mediate smell sensations from air con- papillae of the tongue (60, 61). The association of VEGs taining chemical compounds (81). Another interesting with taste buds of circumvallate and foliate papillae sug- function of salivary lipocalins is that the proteins have gests an interaction with the gustatory function. Human an affinity for actin-binding proteins (79, 82). VEG pro- VEG protein is 18 kD of 176 amino acids, and is found to tein specifically binds to Streptococcus salivarius, just as be homologous to that of rat VEG protein and odorant lipocalin inhibits the growth of S. salivarius (83). VEG binding protein II (OBP II) in rat nasal glands. OBP II in protein inhibits papain activity to a similar extent as do the rat shows a 45.5% amino acid identity in a 178 amino- salivary cystatins (84), suggesting that the acid overlap with the VEG protein (62). Schenkels et al. biophysiological activities of VEG protein lipocalins are (63) reported the biochemical characteristics of human involved in certain antimicrobial processes in the oral lipocalin VEG protein and of extra parotid glycoprotein cavity as well as in ocular tissue. (EP-GP) isolated with hydroxyapatite-binding assay from A new collecting technique from human von Ebner’s the SM and SL glands. EP-GP protein is distributed glands has also been devised to clarify the saliva-taste widely in human tissues in various forms in saliva, sweat, interaction under both stimulated and unstimulated con- tears, nasal mucus, and seminal fluid. EP-GP protein is ditions (85). Using this technique, secreted saliva from identical to secretory actin-binding protein (SABP), a human von Ebner’s glands was analyzed by SDS gel elec- gross cystic disease fluid protein-15 (GCDFP-15) (64-66), trophoresis. The technique also makes it possible to de- a prolactin-binding protein (PIP) (67-69), and a 17-KDa termine VEG specific protein-amino acid sequence and CD4-binding glycoprotein (gp17). Lipocalin is present in the cDNA of lipocalin, as well as to confirm the isolation human tears (70, 71). VEG protein is also identical to tear- and purification of the protein from several species (68, specific pre-albumin (TSPA) based on its amino acid se- 86-88). quences (72-74). Recently, it was found that the proteins Tick salivary gland proteins and toxins belong to from nasal mucous and sweat proteins are biochemically the lipocalin family. Rhodnius prolixus is a Henaiptera identical to VEG proteins and that the lacrimal gland that feeds on living vertebrate blood, and tick salivary shows a VEG protein reaction immunohistochemically. gland proteins (TSGPs) are termed Rhodnius prolixus Immunohistochemical observations of GCDEP-15 protein aggregation inhibitor 1 (RPAI-1), a 19 KD protein. This in the breast have confirmed that VEG protein is synthe- protein shows a to lipocalin, pallidipin sized in the Golgi apparatus. It is present in cytoplasmic and triabin from Triatoma pallidipennis. It has been re- granules in the breast epithelium and apocrin gland, and ported that RPAI-1 is also a member of lipocain family is secreted by exocytosis (64, 75, 76). Cellular localization that inhibits platelet aggregation (89). TSGPs of the soft of VEG protein lipocalin in von Ebner’s gland has shown tick from Ornithodoros savignyi consist of 4 abundant that the protein is found in acinar cells and also on the proteins that play a biological role in salivary gland-gran- cleft side of circumvallate and foliate papillae (77). Im- ules. They have been assigned to be the lipocalin family munohistochemical detection of GCDFP-15 (a lipocain), as well-known tick toxins. TSGP2 and TSGP4 show a high using monoclonal antibodies (GCDFP-15 and D6) in par- sequence identity and an inhibitor of collagen-induced affin sections in salivary gland tumors, has demonstrated platelet aggregation obtained from O. moubata and they that it exists in a higher level in benign tumor epithelia are found to be nontoxic in salivary glands (90). Potent as in pleomorphic adenomas and Warthin’s tumors (78). pharmacological peptides of the lipocalin family have also 6 Mori et al. Antimicrobial peptides in saliva been produced from Rhodnius salivary glands (91). A is found to be elevated in many malignant tumors (101). major salivary allergen (20 KD protein), a member of the The coexpression of Cg and S100A6 in proacinar cells of lipocalin family, has been isolated from insect salivary the rat developing salivary gland may be mediated by glands as procalin (92). Biologically-active peptides of cell migration and proliferation during the postnatally- salivary gland origin in the human and animal kingdom developing stage in acinar cells. have been shown to have a wide range of functions as Endocrine cells have been found to exist in the gas- growth factors, with antimicrobial and hormone-like prop- trointestinal organs with entero-chromaffin cells of the erties based on structural specificity as well as evolution- small intestine and to show high immunoreactivity to Cgs. ary development. In addition, secretory cells of entero-glucagon and gas- trin also show Cg reactivity. Although endocrine cells in Chromogranin the salivary glands have not been described, Cg positive Chromogranins and secretogranins belong to the cells in salivary glands may exist inside digestive entero- granin family. The 3 members of the family, chromogranin chromaffin cells. While normal salivary glands are de- A (CgA), chromogranin B (CgB), and secretogranin void of Cg immunoreactivity, carcinoma of the major sali- II(SgII), are secretory proteins in endocrine chromaffin vary glands has revealed Cgs in 3 cases out of a total of cells, parathyroid cells and adenohypophyseal cells. Three 11 primary small-cell neuroendocrine carcinoma patients other acidic secretory proteins, 1 B1075 gene product (102). (secretogranin III, Sg III), HISL-19 antigen (secretogranin Cgs bind intragranular calcium with a weak affin- VI, Sg VI), and 7B2 (secretogranin V, Sg V) have also been ity and binding is influenced by Mg2 ＋, pH, and ionic discovered (93). Endocrine tissues, parathyroid gland, strength. Electrostatic interactions between soluble acidic thyroid gland, endocrine pancreas, pituitary gland, and proteins and ATP have been detected by nuclear mag- digestive tube non-nerve tissue have been reported to netic resonance in chromaffin granules. Similar interac- contain CgrA cells (94, 95). Cg was first detected by im- tions between Cgs and catecholamines are known and munohistochemistry in salivary glands (96). However, the thus both substances are usually found to coexist. CgA biological role of Cg in salivary glands is unknown. Cgs mimics the inhibitory effect of vasostatins on the contrac- are stored in vesicles in the sympathetic nervous system tion induced in the absence of extracellular calcium by and the adrenal medullary cells, from which they are re- high potassium, but not by endothelin-1 (103). Secretory- leased with catecholamines (97). Cgs have also been de- granule is biosynthesized through the following key steps: tected immunohistochemically in proacinar cells and/or i) calcium signaling mediates the sorting of the granin undifferentiated acinar cells of rat SM gland from the and low pH-induces aggregation in the trans-Golgi net- 1st to 14th postnatal day. An electron microcopic study work; and ii) the aggregations of CgA, CgB and Sg II are showed Cg- positive cells in terminal tubule cells and promoted by millimolar concentrations of Ca2＋and acidic proacinar cells (98). It has recently been found that Cg is pH. These aggregates exclude the secretory proteins, i.e., one type Ca-binding protein and that calmodulin binds albumin, α1-antitrypsin, immunoglobulin and transfer- to Cg via Ca regulation. Acinar cells in mature rat SM rin, and Cgs are retained in the lumen of the TGN after glands are devoid of Cg immunoreactivity, and the granu- perforation of the Gogi membrane in the presence of Ca2＋ lar convoluted segments are also negative for Cg. How- (10 mM) at pH 6.4 (93). The intracellular and extracellu- ever, hypertrophic acinar cells of rat SM gland treated lar processing of CgA has also been involved in the pro- with isoproterenol revealed positive Cg staining. It is sug- teolytic degradation mechanism. CgA processing occurs gested that the expression of Cg in developing SM gland in the extracellular space after release, generating new is involved in cell migration and in differentiation of the and shorter fragments (104). It has recently been reported postnatal stage, and that Cg-positive hypertrophic aci- that bovine vasostatin-1 and bovine CgA exhibit both nar cells treated with isopreternol is mediated via Ca- antibacterial and antifungal activities, and recombinant regulation by isoproternol (99). Postnally-developing sali- and synthetic peptides corresponding to vasostatin also vary glands showed S100A1 and S100A6 immunoreac- have the same activities. The C-terminal moiety of tivity, and S100A6 staining persisted in proacinar cells vasostatin-1 is essential for antifungal activity. from 3 days to 2 weeks with the coexistence of Cg reactiv- Vasostatin-1 is stored in the endocrine cells, as is CgA, ity. S-100A1 expression is confined to pillar and transi- and is liberated from stimulated chromaffin granules and tional cells in the granular and striated ducts from the immune cells following stress. The findings may indicate age of 4 weeks to maturity (100). Although the biological that vasostatin is a new active component in innate im- roles of S100A6 are not well known, it is expressed in a munity (105). Glycosylated and phosphorylated CgA-de- cell cycle-dependent manner in human diploid fibroblasts, rived peptide (bovine adrenal) show antibacterial activ- with its highest expression found in patients with chronic ity (106) and CgB-derived peptide, secretolytin, also ex- myelogenous leukemia as well as its prognostic marker. hibits antibacterial activity (107). Further, the C termi- S100A6, termed calcyclin, is a Zn2 ＋ binding protein and nal of proenkephalin-A-derived peptide possesses anti- Oral Med Pathol 11 (2006) 7 bacterial potency (108). These antimicrobial activities in The distribution of calprotectin in oral squamous the granin derivatives of salivary glands and saliva are epithelium has been detected immunohistochemically considered to act as part of a defense system for prevent- using monoclonal antibody MAC387. The results showed ing oral mucosal infections. that calprotectin was expressed in upper spinous cell lay- ers, while basal and keratinized layers were unreactive. Calprotectin Carprotectin is expressed in oral keratinocytes from nor- Calprotectin is a 36.5-KD protein consisting of 2 mal and non-inflamed oral mucosa, while heavy chains and 1 light chain that are noncovalently orthokeratinized sites including the attached gingiva and linked. Calprotectin comprises a family of novel calcium- hard plate express low levels of calprotectin with a re- binding S100 A proteins: S100A8 (GAG A) and S100A9 stricted pattern (117). Oral epithelial lesions, such as li- (GAG B). S100A8, previously called as GAG A (calgranulin chen planus, candidiasis, herpes stomatitis, and oral hairy A), is synonymous with MRP8 (macrophage migration leukoplakia, are associated with significantly-enhanced inhibitory factor-related protein), and S100A9, previously level of calprotectin compared to that of normal epithe- termed as GAG B (calgranulin B), is synonymous with lium (118). Calprotectin mRNA and protein in cultured MRP14. Historical aspects of the nomenclature and gingival keratinocytes are increased upon treatment with chemical properties of calprotectin have been reviewed lipopolysaccharide (LPS) or interleukin-1 β (IL-1β) as by Johne et al. (109). Miyasaki (110) has reported the cru- well as during inflammation of gingival epithelial cells. cial role of neutrophil for the control of potentially- Thus, calprotectin would be upregulated by inflamma- periodontopathic microorganisms. Neutrophils contain a tory cytokines (119). number of antimicrobial components, including neutro- Calprotectin has been detected in peripheral or phile-derived antimicrobial agents, such as calprotectin whole areas of dental calculus by immunohistochemical complex, defensins, lysozyme, cofactor-binding protein, and immunoblotting analyses, indicating that calprotectin neutral serine protease, bactericidal permeability increas- originating from leucocytes and macrophage could bind ing protein, myeloperoxidase, and a NADP oxidase sys- to the calculus matrix (120). Levels of calprotectin, IL-I tem. The name, calprotectin, was first proposed due to β, and tumor necrosis factor α (TNFα) in gingival crev- the finding that the protein has calcium-binding and an- icular fluid (GCF) are elevated in patients with periodon- timicrobial properties (111). Calprotectin inhibits the tal diseases (121-125). The calprotectin in GCF originates growth of C. albicans and bacteria in vitro. The L1 anti- from granulocyte and monocytes that have infiltrated into gen (leukocyte protein) existing in subsets of tissue mac- the cervical epithelium and into GCF following inflam- rophages in inflammatory lesions consists of two subunits matory irritation. Calprotectin levels in body fluids,

(L1 L and L1 H chains). It is identical to the proteins called plasma, saliva, and synovial fluid have been reported to MRP-8 and MRP14. Rugtveit et al. (112) noted that the be elevated markedly under inflammatory conditions such calprotectin level can be used in rejection diagnosis and as infection by Porphyromonas gingivalis. Calprotectin that calprotectin is upregulated by mononuclear cells that can inhibit binding of the bacterium to the epithelial cells have infiltrated into transplanted kidney. It is reported (121, 123, 126, 127). Calprotectin analysis in parotid sa- that S100A8 (MRP8) alone does not have an anticandidal liva, whole saliva, and mucosal transudate has revealed activiy, whereas a combination of S100A8 and S100A9 that the protein level of parotid saliva is lower than that (MRP14) exhibits significant candidastatic activity (113). of whole saliva or mucosal transudate (128). This finding Muller et al. (114) reported that the calprotectin level in indicates that pure saliva without contamination from whole saliva is considerably higher because the protein desquamated epithelial cells or GCF will show low or trace is secreted by oral epithelium, the inflammatory foci of amounts of calprotectin. It has been reported that the granulocytes and macrophages, and is also leaked from output of calprotectin, as well as that of IgA, IgG and the gingival crevices. Kleinegger, Stoeckel and Kurago albumin in whole saliva, is lower in Sjögren’s syndrome (115) reported that salivary calprotectin levels in patients (SS) than in a control. In spite of the reduced output of with or without candidiasis are positively correlated with saliva per minute, the markedly increased concentrations the intensity of oral candidal infection. A high concentra- of calprotectin in whole saliva are observed in primary tion of calprotectin is found in plasma under conditions SS patients (129). Salivary calprotectin levels correlate of chronic inflammation as a result of the protein release significantly with plasma calprotectin levels and with and turnover of leucocytes. C5a is a potent proinfla- several ocular variables, but only weakly with salivary mmatory mediator that exerts its anaphylatoxic activi- flow and serum rheumatoid factor, and not at all in ties by binding to specific C5a receptors on polymorpho- sialadenitis. Thus, salivary calprotectin levels would be nuclear neutrophils, monocytes and non-mycloid cells. The associated with SS glandular pathology (130). Oral can- chemotaxins C5a and formyl peptide (formyl-methionine- didiasis is one of the common fungal infections in den- leucine phenylalanine: fMLP) have been shown to ture-associated mucositis, and is also associated with oral stimutate the release of calprotectin (116). mucosal leukoplakia as a precancerous condition. In ad- 8 Mori et al. Antimicrobial peptides in saliva dition, it has been asserted that candidal infection is par- tissue (156). In mucoceles, hBD-1 is also expressed at the ticularly associated with human immunodeficiency virus luminal site of a duct-like structure. However, hBD-1 in (HIV) infection (131, 132). Challacombe (131) classified saliva is undetectable by dot-blot assay. This may be due candidal infection in oral mucosa into 6 classes based on to the masking effect of mucin. Western blotting of whole clinical features and causes, and showed that nonspecific saliva has clarified a single band of hBD-1, indicating immunity, serum salivary antibodies, and cell-mediated that salivary hBD-1 is less processed than that present immunity could be considered as associated factors. in urine (145). Other origins of (hBD-2) in hu- Greenspan (132) noted that, among HIV patients, the mans are gingival tissue (143), plasma (157) and oral prevalence of oral candidiasis ranged from 9% to over 90% epithelium (158). Abiko et al. (159) investigated the lo- depending on the stage of their infection. Superficial in- calization of hBD-2 in normal oral epithelium and epi- fection in oral mucosa by Candida occurs early in the thelial lesions (carcinoma and leukoplakia). The results disease, and the interaction of anticandidal calprotectin clearly showed that hBD-2 expression is more intense in with keratinocytes should always be evaluated. High hyperkeratinized than in ortho- or non-keratinized epi- calprotectin levels are found in subjects with candidiasis, thelium. In the oral epithelium of lichen planus (n=30), and its concentration correlates positively with the de- strong hBD-2 expression was found in 13 cases in the gree of candidal infections (114, 115). Calprotectin is com- spinous and parabasal layers, and positive staining was prised of calcium-binding S100 proteins that bind to cal- also seen in the remaining 17 cases from the granular to cium as well as to zinc. Matrix metalloproteinases, a fam- the keratinized layer. The expression of oral epithelial ily of zinc-dependent enzymes, are inhibited by hBD-2 is divided into two types: the wide staining type, calprotectin (133). The growth inhibitory effect of such as peptide, is distributed from the parabasal to the calprotectin on Candida is reversed by micromolar quan- spinous cells, while the limited staining type is distrib- tities of zinc. These findings suggest that calprotectin in- uted from the granular to the keratinized cells (160, 161). hibits candidal growth through competition for zinc (134, hBD-2 is specificially induced by bacterial infections and 135). inflammatory cytokines in the oral mucosa. Human gin- gival epithelial cells express hBD-1 and hBD-2 as well as Defensins cytokines and chemokines. hBD-2 mRNA is induced by Human defensins are cationic antimicrobial pep- the cell wall extract of Fusobacterium nucleatum, but not tides that are divided into α and β defensins. α - by that of P. gingivalis. In addition, hBD-2 mRNA is in- Defensins consist of 6 types: 1-4 α -defensins (HNP 1-4) duced by TNF- α and phorbol myristrate acetate. Stud- are contained in neutrophils (136) and show non-oxida- ies on the inhibitors of hBD-2 have shown that stimula- tive antimicrobial activity in phagocytic vesicles, while 5 tion of hBD-2 mRNA by F. nucleatum requires both a new and 6 α -defensins (HNP 5, 6) are found in the Paneth gene transcription and a new protein synthesis, indicat- cells of the intestine (137). On the other hand, β - ing the possibility that F. nucleatum might play a role in defensins (hBD-1 and -2) are found in intestinal epithe- stimulating mucosal epithelial cells to maintain the bar- lium (138). In mice, the β-defensin 1 (mBD-1) gene is rier involved in homeostasis and host defense (162). Cor- localized at the proximal portion of 8 and related expression of hBD-1, -2, -3 mRNA and inflamma- mBD-1 is detected in epithelia of the lung and urogenital tory cytokines has been clarified. The expression of TNF- tract (139, 140). hBD-2 and β-defensin 3 (hBD-3) are pro- αmRNA is correlated with hBD-1 mRNA. Furthermore, duced in the skin (141) and upregulated in vulgaris (142). the correlation between TNF- α mRNA and hBD-2 or Defensins are also present in oral tissues (143-148) as hBD-3 mRNA is much higher than that between TNF-α well as in nasal (149, 150) and tracheal epithelium (151, and hBD-1 mRNA in young children (163). While 31 types 152). Alpha and β defensins differ in their primary se- of hBDs have recently been recognized by computational quences (153). HNP1-3 transcripts are evident in the SM search (164), molecular and genetic investigations of hBDs gland, prostate, placenta and trachea. The SM gland may clarify genetic risks involved in oral infections. High shows a high concentration of hBD-1 and the relative extracellular Ca2+ concentration and thapsigargin could amounts of the protein in trachea and prostate are both increase hBD-2 gene expression in oral epithelial cells, 77% versus SM gland (154). and this enhancement is dependent on the elevation of Immunohistochemical observations have revealed intercellular Ca2+. The interaction between F. nucleatum that the ductal segment is a source of hBD-1 and that and the epithelial surface also activates hBD-2 via a cal- hBD-1 is localized in the luminal site of the excretory cium-dependent pathway (165). duct of the SM gland. It increases in the inflammatory Dale and Krisanaprakornkit (153) have docu- infiltrates distributed beneath the ducts. Keratinized re- mented the role of β-defensins as a part of the innate gions of pleomorphic adenoma express positive staining immune system. The contribution of human HNP 1- 3 to for hBD-2 with various intensities (155); however, hBD-2 the anti-HIV-1 activity of CD8 antiviral factor has also mRNA has been detected in few samples of salivary gland been estimated (166). Oral Med Pathol 11 (2006) 9

Salivary HNP 1 increases slightly whereas hBD-1 salivary gland tumors. LF reactivity in human fetal sali- and 2 decrease in patients with candidiasis (167). In con- vary glands is demonstrated in serous acinar cells (25 w trast, α -defensins in saliva from patients with lichen to 40 w fetus) and in intercalated, striated and excretory planus, leukoplakia and grositis are increased about 10- ducts (25 w to 29 or 30 w) (186). fold compared to healthy controls (168). It has recently α-ACT and α-AT as proteinase inhibitor might been found that murine β -DF 2β acts directly on im- have biochemical functions against chymotrypsin, trypsin, mature dendritic cells as an endogeneous ligand for Toll- elastase, collagenase and plasmin. They are expressed in like receptor 4 and this induces the up-regulation of wound healing processes after tissue damage as well as costimulatory molecules and dendritic cell maturation in tissue remodeling. Alpha-ACT and α-AT are found in (169). The overexpression of α-defensins (human neu- many tumors and body fluids such as saliva, tears, milk, trophil peptides HNP 1, HNP 2, HNP 3) has been reported amniotic fluid and semen, as well as duodenal bile (194- in squamous cell carcinoma of the tongue compared with 201). Human salivary glands are reactive to α1-AT in autogenous non-tumor tissue. The result is attributable ductal segments but unreactive to α-ACT in glandular to oncolytic activity in neutrophil infiltration (170). components (202). In human fetal glands, α-ACT is Mizukawa et al. (171) reported that human α-defensins present in serous cells (23-34 w) and in ducts (15-20 w), are expressed in the SM glands of oral carcinoma patients. while α-AT is expressed in both serous and mucous cells Significantly higher levels of hBD-3 mRNA in healthy (20-40 w) and ducts (15-40 w) at strong level (186). Sali- gingiva have recently been reported, indicating the im- vary pleomorphic adenoma and other tumors are found portance of this protein in the protection of periodontal to show varying reactivities to α-ACT and α-AT (202). tissues (172, 173). Discussion

Lysozyme, Lactoferrin, α1-antichymotrypsin and α1-an- Human whole saliva contains many peptides se- titrypsin creted from the salivary glands or from other sources, as In addition to recently detected and characterized described above. These peptides show antimicrobial ac- peptides, it has been well documented that salivary glands tivity and/or unique characteristics that contribute to the secret antibacterial substances such as lysozyme (LY), defense system of the oral cavity. These peptides have lactoferrin (LF), and anti-proteolytic proteins ( α1- been detected using specific antibodies in the normal antichymotrypsin: α-ACT; α1-antitrypsin: α-AT) (174). major and minor salivary glands, as well as in obstruc- They are present not only in salivary glands (175-179) tive lesions and salivary glands tumors. It is therefore but also in sweat (180) and nasal glands (181), and in the expected that such peptides and/or their synthetic ana- digestive tract (182). It is considered that these antibac- logues could be utilized in maintaining oral health and terial and antiproteolytic enzymes also contribute towards prophylaxsis of oral diseases (203, 204). Antimicrobial ac- the defense system of the oral cavity. tivities of these proteins are enhanced by forming com- LY was found in human secreted fluids by Fleming plexes with salivary mucin (205, 206) or by combining in 1922. LY in giant cells of giant cell tumors and in leu- with each other (207). Immunogloblin A and kocytes and exfoliated oral epithelial cells has also been immunogloblin isotypes are secreted mainly from minor reported (183, 184). LY is a neuraminidase and can de- salivary glands (208, 209). These antimicrobial peptides grade neuraminic acid in bacterial cell walls, and thus are also detected in the gastrointestinal tract as well as has bacteriolytic properties. Localization of LY in sali- in the upper respiratory epithelia, indicating their roles vary glands has been investigated with an immunohis- against microbial infections in such regions. Pleomorphic tochemical method, which demonstrated that ductal cells adenoma shows prominent reactivities toward these an- are strongly positive and serous acinar cells are occasion- timicrobials, which may enable this tumor to gradually ally reactive. Human fetal salivary glands expressed LY enlarge without triggering infectious processes (174). Al- in acinar cells (usually serous type), and in intercalated though salivary gland-secreted substances have attracted duct cells in specimens from 21, 23 to 40 weeks (185, 186). much attention, recent advances in oral sciences have led Salivary pleomorphic adenoma also shows positive reac- to the isolation and characterization of many novel anti- tion for LY and LF. microbial peptides in whole saliva. A recent study has LF is an ion-binding protein detected in human evaluated the entire issue of whole saliva as a defender milk, which has antibacterial activity because it removes of the oral cavity (210). The origins of these peptides in- bacterial iron. Synthesis and subcellular localization of clude not only salivary glands, but also keratinocytes, leu- LF in salivary gland cells have been detected. This indi- kocytes and monocytes. PLUNC (palate, lung, and nasal cates the secretory mechanism of LF by acinar cells (185, epithelium clone)-like proteins have been detected, and 186). LF is expressed in both normal salivary gland cells it was found that they relate to the host defence protein and tumors cells (187-193). In these studies, the BPI (bactericidal/permeability-increasing protein). The coexpression of LF with LY has been demonstrated in biological roles of PLUNC-like proteins in the interface 10 Mori et al. Antimicrobial peptides in saliva between host tissue and pathogenic microenvironment tion of antimicrobial peptides with the biological mem- have been reported (211). Synthetic peptides with anti- brane of bacteria and the antibacterial mechanism of microbial activty have also been reported. Cationic pep- peptides have been reviewed (215, 216). tides, Dhvar 4 ( α-helical peptides) active against C. A number of antimicrobial peptides synthesized by albicans, have been synthesized (JH8194, JH8195, and multicellular organisms are encoded by the genome and JH8944). Among them, JH8194 exhibits the most potent are biosynthesized by the regular processes of gene tran- fungicidal activity (212). scription and ribosomal translation, followed by the pro- Tenovuo (204) devised a clinically effective method teolytic processing of the gene product. This, in turn, would against xerostomia by applying antimicrobial host pro- facilitate the exploration of the exocrine salivary secre- teins. Recently, Huang (213) reported that the levels of tion of transgene products and their possible clinical ap- 10 proteins in whole saliva were significantly different plication to therapeutic uses and the maintenance of oral when bleeding had occurred in the oral cavity. The 10 health. Further planning is underway to evaluate pos- proteins include α-1-anitrypsin, apolipoprotein A-1, sible and continuing processes in the maintainance of oral cystatin A, SA, SA-III, and SN, enolase I, hemoglobulin health using new technical approaches such as gene tar- β-chain, thioredoxin peroxiredoxin B, and a prolactin- geting of those antimicrobial peptides in major salivary inducible protein. These findings seem to indicate that glands; experimental studies using human subjects are antimicrobial peptides could be used as prophylactic and already in progress (217). therapeutic agents against oral diseases (especially in- It is well documented that the oral cavities of hos- fections and xerostomia) and that a quantitative alter- pitalized patients and elderly nursing home residents are ation in peptides can have a diagnostic significance. frequently infected with multidrug-resistant microorgan- In considering the utilization of antimicrobial pep- isms, such as methicillin-resistant Staphylococcus aureus, tides, clarification of the mechenism(s) by which they ex- vancomycin-resistant enterococci, and C. albicans. In- hibit antimicrobial activities under a variety of oral envi- creasing evidence has revealed that the accumulation and ronmental conditions would be a crucial issue. Increas- disturbance of oral flora and the colonization of opportu- ing findings concerning antimicrobial mechanisms dem- nistic pathogens in the oral cavity and/or the oropharyn- onstrate that antimicrobial peptides are characterized by geal tract are key factors that increase the risk of devel- their action on the lipid matrix of the microbial mem- oping pneumonia via silent aspiration in immuno-com- branes. Membrane-acting antimicrobial peptides are promised hosts (218-220). This condition occurs rapidly translocated across the membrane by forming pores, and as a result of the use of cytotoxic drugs and broad-spec- their common structural specificity is comprised of 15-40 trum antibiotics, decreased salivary flow, changes in sali- amino residues. In the amino acid composition of antimi- vary antibody levels, and the damage of the epithelial crobial peptides, the presence of the cationic amino acids cells. The membrane-acting antimicrobial peptides may is essential. This suggests that they have an ability to also contribute to the prevention of such infections, in- associate with membrane lipids in the bilayer, as well as stead of conventional antibiotic therapy. Thus, investiga- effects of membrane-association on the bilayer structure tions concerning the effects of antimicrobial peptides in the cell wall. Polycationic molecules bind to the outer found in saliva and salivary glands against not only com- membrane that possesses negatively-charged lipopolysac- mensal oral microorganisms but also against exogenous charide molecules in Gram-negative bacteria. Thus, they pathogens would provide an important social benefit. would be able to alter the physical structure of the bi- layer to be permeable for normally- impermeable hydro- Addendum phobic molecules. The killing of bacteria by membrane Recently, Lee (221) reported that a new antimicro- permeabilization appears to be rapid. However, the mul- bial peptide, mucocidin, has been isolated from the exo- tiple mechanisms of action, especially those involving the crine gland. Mucocidin is expressed in mucosal epithelial perturbation of cellular metabolism, have yet to be un- cells and accessory glands, and is secreted into mucosal equivocally determined. Defensin and also ex- fluid and glandular secretions as an active mature pep- ert their effects rapidly on the bacterial wall (214). The tide. Mucocidin encoded by the 527 nucleotide CDNA and structure of all defensins has been found to assume aβ- its synthetic peptide are bactericidal against Escherichia sheet structure, and plant and insect defensins contain coli and S. aureus (including methicillin-resistant S. anÉø-helical structural motif. All defensins have an aureus), and C. albicans. Immunohistochemical and in amphipathic structure that allows them to act on bacte- situ hybridization confirmed that gene expression is rial membranes in a manner similar to that of shorter widely distributed in exocrine glands and secretory ducts. amphipathic peptides. The antimicrobial specificity of Mococidin in oral epithelia provides the similar mecha- these peptides would arise from many causes. The differ- nism with protecting mucosal surfaces as already known ences in membrane interactions would result in different antimicrobial peptides. rates of transport of the peptides into cells. The interac- Oral Med Pathol 11 (2006) 11

References 1998; 45: 19-25. 1. Black C, Allan I, Ford SK, et al. Biofilm-specific surface prop- 17. Henskens YMC, Veerman ECI, Mantel MS, et al. Cystatins S erties and protein expression in oral Streptococcus sanguis. and C in human whole saliva and in glandular salivas in Arch Oral Biol 2004; 49: 295-304. periodontal health and disease. J Dent Res 1994; 73: 1606- 2. Okuda K, Kato T and Ishihara K. Involvement of perio- 14. dontopathic biofilm in vascular diseases. Oral Dis 2004; 10: 18. Skaleric U, Babnik J, Curin V, et al. Immunochemical 5-12. quantitation of cysteine proteinase inhibitor cystatin C in 3. Llewellyn CD and Warnakulasuriya S. The impact of stoma- inflamed human gingival. Arch Oral Biol 1989; 34: 301-5. tological disease on oral health-related quality of life. Eur J 19. Aguirre A, Suraweera LA, Banderas JA, et al. Levels of sali- Oral Sci 2003; 111: 297-304. vary cystatins in periodontal healthy and diseased older 4. Mombelli A. Antimicrobial agents in periodontal prevention, adults. Arch Oral Biol 1992; 37: 355-61. therapy and maintenance. Oral Dis 2003; 9: Supple. (1): 71- 20. Henskens YMC, Van der Velden U, Veerman ECI, et al. Pro- 2. tein, albumin and cystatin concentrations in saliva of healthy 5. Henskens YMC, Veerman ECI and Nieuw Amerongen AV. subjects and patients with gingivitis or periodontitis. J Cystatins in health and disease. Biol Chem Hoppe-Seyle 1996; Periodontol 1993; 28: 43-8. 377: 71-86. 21. Tseng CC, Tseng CP, Levine MJ, et al. Differential effect to- 6. Dickinson DP. Cystein peptidases of mammals: their biologi- ward inhibition of papain and cathepsin C by recombinant cal roles and potential effects in the oral cavity and other human salivary cystatin SN and its variants produced by a tissues in health and disease. Crit Rev Oral Biol Med 2002; baculovirus system. Arch Biol Chem Biophy 2000; 380: 133- 13: 238-75. 40. 7. Sabatini LM, Warner TF, Saitoh E, et al. Tissue distribution 22. Lerner UH and Grurb A. Human cystatin C, a cysteine pro- of RNAs for cystatins, histatins, statherin, and proline-rich teinase inhibitor, inhibits bone resorption in vitro stimulated salivary proteins in humans and macaques. J Dent Res 1989; by parathyroid hormone and parathyroid hormone-related 68: 1138-45. peptide of malignancy. J Bone Min Res 1992; 7: 433-40. 8. Isemura S, Saitoh E, Sanada K, et al. Identification of full- 23. Tsushima H, Mine H, Kawakami Y, et al. Candida albicans sized forms of salivary (S-type) cystatins (cystatin SN, aspartic proteinase cleaves and inactivates human epider- cystatin SA, cystatin S, and two phosphorylated forms of mal cysteine proteinase inhibitor, cystatin A. Microbiology cystatin S in human whole saliva and determination of phos- 1994; 140: 167-71. phorylation sites of cystatin S. J Biochem 1991; 110: 648-54. 24. Takahashi M, Tezuka T and Katsumura N. Inhibition of 9. Bedi GS. Amino acid suquence of inducible cysteine protein- growth and cysteine proteinase activity of Staphylococcus ase inhibitor (cystatin) from submandibular glands of iso- aureus V8 by phosphorylated cystatin a in skin cornified proterenol-treated rats. Arch Biochem Biophys 1989; 273: envelope. FEBS Letters 1994; 355: 275-8. 245-53. 25. Freije JP, Balbin M, Abrahamson M, et al. Human cystain D. 10. Bedi GS. The effect of adrenergic agonists and antagonists cDNA cloning, characterization of the Escherichia coli ex- on cysteine-proteinase inhibitor (cystatin) in rat saliva. Arch pressed inhibitor, and identification of the native protein in Oral Biol 1991; 36: 611-8. saliva. J Biol Chem 1993; 268: 15737-44. 11. Shaw PA and Barka T. β-Adrenergic induction of a cysteine- 26. Hall A, Ekiel I, Mason RW, et al. Structural basis for differ- proteinase inhibitor mRNA in rat salivary glands. Biochem ent inhibitory specificities of human cystatins C and D. Bio- J 1989; 257: 685-9. chemistry 1998; 37: 4071-9. 12. Naito Y, Suzuki I and Hasegawa S. Induction of cystatin S in 27. Lindahl P, Abrahamson M and Bjork I. Interaction of recom- rat submandibular glands by papain. Arch Oral Biol 1992; binant human cystatin C with the cysteine proteinases pa- 37: 861-5. pain and actinidin. Biochem J 1992; 281: 49-55. 13. Barka T and Van der Noen H. Expression of the for 28. Keppler D, Waridel P, Abrahamson M, et al. Latency of cathe- cysteine proteinase inhibitors cystatins in rat submandibu- psin B secreted by human colon carcinoma cells is not linked lar salivary glands. Arch Oral Biol 1994; 39: 307-14. to secretion of cystatin C and is relieved by neutrophil 14. Shaw PA, Cox JL, Barka T, et al. Cloning and sequencing of elastase. Biochim Biophys Acta 1994; 1226: 117-25. cDNA encoding a rat salivary cysteine proteinase inhibitor 29. Burnett D, Abrahamson M, Devalia JL, et al. Synthesis and inducible by β-adrenergic agonists. J Biol Chem 1988; 263: secretion of procathepsin B and cystatin C by human bron- 18133-7. chial epithelial cells in vitro: modulation of cathepsin B ac- 15. Yamada K. Immunohistochemical studies of large mobile tivity by neutrophil elastase. Arch Biochem Biophys 1995; protein in rat salivary glands. J Jpn Stomatol Soc 1992: 41: 317: 305-10. 52-65. 30. Vigneswaran N, Wu J and Zacharias W. Upregulation of 16. Yamada K, Letic-Gavrilovie A, Mori M, et al. Immunohis- cystatin M during the progression of oropharyngeal squa- tochemical and quantitative analysis of cystatin S in rat sub- mous cell cartinoma from primary tumor to metastasis. Oral mandibular and sublingual glands of rats. Serb J Stomatol Oncol 2003; 39: 559-68. 12 Mori et al. Antimicrobial peptides in saliva

31. Oppenheim FG, Xu T, McMillian FM, et al. Histatins, a novel 2004; 49: 11-22. family of histidine-rich proteins in human parotid secretion. 48. Takano K, Malamud D, Bennick A, et al. Localization of sali- Isolation, characterization, primary structure, and fungistatic vary proteins in granules of human parotid and subman- effects on Candida albicans. J Biol Chem 1988; 263: 7472-7. dibular acinar cells. Crit Rev Oral Bio Med 48; 4: 399-405. 32. Troxler RF, Offner GD, Xu T, et al. Structural relationship 49. Shrestha JP, Hashimoto J, Takagi H, et al. Immunoreactive between human salivary histatins. J Dent Res 1990; 69: 2-6. histatin 5 in salivary gland tumors. Acta Histochem Cytochem 33. Xu T, Levitz SM, Diamond RD, et al. Anticandidal activity of 1994; 27: 527-34. major human salivary histatins. Infect Immun 1991; 59: 2549- 50. Li J, Helmerhorst EJ, Yao Y, et al. Statherin is an in vivo 54. pellicle constituent: identification and immuno-quantifica- 34. Edgerton M, Koshlukova SE, Araujo MW, et al. Salivary tion. Arch Oral Biol 2004; 49: 379-85. histatin 5 and human neutrophil defensin 1 kill Candida 51. Schlesinger DH and Hay DI. Complete covalent structure of albicans via shared pathways. Antimicrob Agents Chemother statherin, a tyrosin-rich acidic peptide which inhibits cal- 2000; 44: 3310-6. cium phosphate precipitation from human parotid saliva. 35. Bercier JA, Al-Hashimi I, Haghighat N, et al. Salivary J Biol Chem 1997; 252: 1689-95. histatins in patients with recurrent oral candidiasis. J Oral 52. Jensen JL, Lamkin MS, Troxler RF, et al. Multiple forms of Pathol Med 1999; 28: 26-9. statherin in human salivary secretions. Arch Oral Boil 1991; 36. Helmerhorst EJ, Breeuwer P, van’t Hof W, et al. The cellular 36: 529-34. target of histatin 5 on Candida albicans is the energized 53. Douglas WH, Reeh ES, Ramasubbu N, et al. Statherin: a major mitochondrion. J Biol Chem 1999; 274: 7286-91. boundary lubricant of human saliva. Biochem Biophys Res 37. Imatani T, Kato T, Minaguchi K, et al. Histatin 5 inhibits Comm 1991; 180: 91-7. inflammatory cytokine induction from human gingival fibro- 54. Schwartz SS, Hay DI and Schluckebier SK. Inhibition of cal- blasts by Porphyromonas gingivalis. Oral Microbiol Immunol cium phosphate precipitation by human salivary statherin: 2000; 15: 378-82. structure-activity relationships. Calcif Tissue Int 1992; 50: 38. Gusman H, Leon C, Helmerhorst EJ, et al. Salivary histatin 511-7. 5 is an inhibitor of both host bacterial enzymes implicated 55. Hay DI, Smith DJ, Schluckebier SK, et al. Relationship be- in periodontal disease. Infect Immun 2001; 69: 1402-8. tween concentration of human salivary statherin and inhi- 39. Richardson DF, Johnson M, Raj PA, et al. The influence of bition of calcium phosphate precipitation in stimulated hu- histatin-5 fragments on the mineralization of hydroxyapa- man parotid saliva. J Dent Res 1984; 63: 857-63. tite. Arch Oral Biol 1993; 38: 997-1002. 56. Jensen JL, Xu T, Lamkin MS, et al. Physiological regulation 40. Yin A, Margolis HC, Grogan J, et al. Physical parameters of of the secretion of histatins and statherins in human pa- hydroxyapatite adsorption and effect on candicidal activity rotid saliva. J Dent Res 1994; 73: 1811-7. of histatins. Arch Oral Biol 2003; 48: 361-8. 57. Contucci AM, Inzitari R, Agostino S, et al. Statherin levels in 41. Helmerhorst EJ, Van’t Hof W, Veerman ECI, et al. Synthetic saliva of patients with precancerous and cancerous lesions histatin analogues with broad-spectrum antimicrobial ac- of the oral cavity: a preliminary report. Oral Dis 2005; 11: tivity. Biochem J 1997; 326: 39-45. 95-9. 42. Driscoll J, Zuo Y, Xu T, et al. Functional comparison of native 58. Raji PA, Jhonsson M, Levine MJ, et al. Dependence on se- and recombinant human salivary histatin 1. J Dent Res 1995; quence, charge, hydrogen bonding potency, and helical con- 74: 1837-44. formation for adsorption to hydroxyapatite and inhibition of 43. Helmerhorst EJ, Hodgson R, van’t Hof W, et al. The effects of mineralization. J Biol Chem 2002; 267: 5968-76. histatin-derived basic antimicrobial peptides on oral biofilms. 59. Blaker M, Kock K, Ahlers C, et al. Molecular cloning of hu- J Dent Res 1999; 78: 1245-50. man von Ebner’s gland protein, a member of the lipocalin 44. Iontcheva I, Oppenheim FG and Troxler FG. Human sali- superfamily highly expressed in lingual salivary glands. vary mucin MG1 selectively forms heterotypic complexes Biochim Biophys Acta 1993; 1172: 131-7. with amylase, proline-rich proteins, statherin, and histatins. 60. Kock K, Blaker M and Schmale H. Postnatal development of J Dent Res 1997; 76: 734-43. von Ebner’s glands: accumulation of a protein of the lipocalin 45. Edgerton M, Koshlukova SE, Lo TE, et al. Candicidal activ- superfamily in taste papillae of rat tongue. Cell Tissue Res ity of salivary histatins, identification of a histatin 5-bind- 1992; 267: 313-20. ing protein on Candida albicans. J Biol Chem 1998; 273: 61. Kock K, Ahlers C and Schmale H. Structural organization of 20436-47. the genes for rat von Ebner’s gland proteins 1 and 2 reveals 46. Gusman H, Lendenmann U, Grogan J, et al. Is salivary their close relationship to lipocalins. Eur J Biochem 1994; histatin 5 a metallopeptide? Biochim Biophys Acta 2001; 221: 905-16. 1545: 86-95. 62. Dear TN, Campbell K and Rabbitts TH. Molecular cloning of 47. Gusman H, Leone C, Helmerhorst EJ, et al. Human salivary putative odorant-binding and odorant-metabolizing proteins. gland-specific daily variations in histatin concentrations Biochemistry 1991; 30: 10376-82. determined by a novel quantitation technique. Arch Oral Biol 63. Schenkels CPM, Veerman CI and Amerongen VN. EP-GP and Oral Med Pathol 11 (2006) 13

the lipocalinnVEGh, two different human salivary 20-kDa properties. Biol Chem Hoppe-Seyler 1994; 375: 609-15. proteins. J Dent Res 1995; 74: 1543-50. 80. Iwasaki S, Aoyagi H and Yoshizawa H. Immunohistochemi- 64. Mazoujian G, Pinkus GS, Davis S, et al. Immunohistochem- cal detection of the expression of keratin 14 in the lingual istry of a gross cystic disease fluid protein (GCDFP-15) of epithelium of rats during the morphogenesis of filiform pa- the breast. A marker of apocrine epithelium and breast pillae. Arch Oral Biol 2003; 48: 605-13. cartinomas with apocrine features. Am J Pathol 1983; 81. Flower DR. The lipocalin protein family: a role in cell regula- 110:105-12. tion. FEBS Letter 1994; 354: 7-11. 65. Haagensen DE Jr, Dilley WG, Mazoujian G, et al. Review of 82. Schaller J, Akiyama K, Kimura H, et al. Primary structure of GCDFP-15, an apocrine marker protein. Ann NY Acad Sci a new actin-binding protein from human seminal plasma. 1990; 586: 161-77. Eur J Biochem 1991; 196: 743-50. 66. Myal Y, Iwasiow B, Yarmill A, et al. Tissue-specific androgen- 83. Schenkels LCPM, Ligtenberg AJM, Veerman ECI, et al. In- inhibited gene expression of a submaxillary gland protein/ teraction of the salivary glycoprotein EP-GP with the bacte- GCDFP-15 gene. Endocrinol 1994; 135: 1605-10. rium of Streptococcus salivarius. J Dent Res 1993; 72: 1559- 67. Shiu RO and Iwasiow BM. Prolactin-inducible proteins in 65. human breast cancer cells. J. Biol Chem 1985; 260: 11307- 84. van’t Hof W, Blankenvoorde MFG, Veerman ECI, et al. The 13. salivary lipocalin von Ebner’s gland protein is a cysteine 68. Murphy LC, Tsuyuki D, Myal Y, et al. Isolation and sequenc- proteinase inhibitor. J Biol Chem 1997; 273: 1837-41. ing of a cDNA clone for a prolactin-indusible protein (PIP): 85. Spielman AI, D’Abundo A, Field RB, et al. Protein analysis of regulation of PIP gene expression in the human breast can- human von Ebner saliva and a method for its collection from cer cell line, T-47D. J Biol Chem 1987; 262: 15236-41. the foliate papillae. J Dent Res 1993; 72: 1331-5. 69. Myal Y, Gregory C, Wang H, et al. The gene for prolactin- 86. Windass JD, Mullins JJ, Beecroft LJ, et al. Molecular cloning inducible protein (PIP), uniquely expressed in exocrine or- of cDNA/2 mouse submaxillary glands. Nucleic Acid Res 1984; gans, maps to chromosome 7. Somat Cell Mol Genet 1989; 12: 1361-76. 15: 265-70. 87. Lassagne H, Ressot C, Mattei MG, et al. Assignment of the 70. Gachon AM . Lipocalins: Do we taste with our tears? Trends human tear gene (LCN1) to 9q34 by in situ hybridization. Biochem Sci 1993; 18: 206-7. Genomics 1993; 18: 160-1. 71. Glagow BJ, Heinzmann C, Kojis T, et al. Assignment of tear 88. Holzfeind P and Redl B. Structural organization of the gene lipocalin gene to human chromosome 9q34-9qter. Curr Eye encoding the human lipocalin in Escherichia coli. Gene 1994; Res 1993; 12: 1019-23. 139: 177-83. 72. Gachon AM, Richard J and Dastugue B. Human tears-nor- 89. Francischetti IM, Ribeiro JM, Champagne D, et al. Purifica- mal-protein pattern and individual protein determinators tion, cloning, expression, and mechanism of action of a novel in adults. Curr Eye Res 1982; 5: 301-8. platelet aggregation inhibitor from the salivary gland of the 73. Janssen PT and Van Bijsterveld OP. The relations between blood-sucking bug Rhodnius Prolixus. J Biol Chem 2000; 275: tear fluid and concentrations of lysozyme, tear-specific 12639-50. prealbumin and lactoferrin. Exp Eye Res 1983; 36: 773-9. 90. Mans BJ, Louw AI and Neitz AW. The major tick salivary 74. Garibotti M, Christiansen H, Schamel H, et al. Porcine VEG gland proteins and toxins from the soft tick, Ornithdoros proteins and tear pre prealbumins. Chem Senses 1995; 20: savignyi, are part of the tick lipocalin family: implications 69-76. for the origins of tick toxicoses. Mol Biol Evol 2003; 20: 1158- 75. Mazoujian G, Warhol MJ and Haagensen DE Jr. The ultra- 67. structural localization of gross cystic disease fluid protein 91. Ribeiro JM, Anderson J, Siva-Neto MA, et al. Exploring the (GCDFP-15) in breast epithelium. Am J Pathol 1984; 116: sialome of the blood-sucking bug Rhodnius prolixus. Insect 305-10. Biochem Mol Biol 2004; 34: 61-79. 76. Pagani A, Eusebi V and Bussolati G. Detection of PIP-GCDFP- 92. Paddock CD, McKerrow JH, Hansell E, et al. Identification, 15 gene expression in apocrine epithelium of the breast and cloning, and recombinant expression of procalin, a major salivary glands. Appl Immunohistochem 1994; 2: 29-35. triatomine allergen. J Immunol 2001; 167: 2694-9. 77. Kock K, Blaker M and Schmale H. Postnatal development of 93. Huttner WB, Gerdes HH and Rosa P. The granin von Ebner’s glands: accumulation of a protein of the lipocalin (chromogranin/secretogranin) family. TIBS 1991; 16: 27-30. superfamily in taste papillae of rat toung. Cell Tissue Res 94. Buffa R, Gini A, Pelagi M, et al. Immunoreactivity of hor- 1992; 267: 313-20. monally-characterized human chromogranin B (Bill and B13) 78. Swanson PE, Pettinato G, Lillemoe TJ, et al. Gross cystic and chromogranin A (AII) monoclonal antibodies. Arch Histol disease fluid protein-15 in salivary gland tumors. Arch Pathol Cytol 1989; 52: 99-105. Lab Med 1991; 115: 158-63. 95. Simon JP and Aunis D. Biochemistry of the chromogranin A 79. Schenkels LCPM, Schaller J, Walgreen-Weterings E, et al. protei family. Biochem J 1989; 262: 1-13. Identify of human extra parotid glycoprotein (EP-GP) with 96. Letic-Garvilovic A, Abe K and Mori M. Chromogranin B-like secretory actin binding protein (SABP) and its biological immunoreactivity in the mouse submandibular salivary 14 Mori et al. Antimicrobial peptides in saliva

gland during postnatal development. Acta Histochem 1990; L1 antigen (Calprotein) by tissue macrophages reflects re- 89: 1-10. cent recruitment from peripheral blood rather than 97. Rindi G, Buffa R, Sessa O, et al. Chromogranin A, B and C upregulation of local synthesis: Implications for rejection immunoreactivity of mammalian endocrine cells. Distribu- diagnosis in formalin-fixed kidney specimens. J Pathol 1996; tion, distinction from costored hormones/prohormones and 180: 194-9. relationship with the argyrophil aomponent of secretory 113. Murphy AR, Lehrer RI, Harwig SSL, et al. In vitro granules. Histochemistry 1986; 85: 19-28. candidastatic properties of the human neutrophil calprotectin 98. Kunikata M. Immunohistochemical expression of complex. J Immunol 1993; 151: 6291-301. chromogranin in rat submandibular gland. J Jpn Stomatol 114. Muller F, Froland SS, Brandtzaeg P, et al. Oral candidiasis Soc 1994; 43: 551-67. is associated with low levels of parotid carplotectin in indi- 99. Kunikata M, Tamada K, Ogata K, et al. Immunohistochemi- viduals with infection due to human immunodeficiency vi- cal expression of chromogranin in postnasal developmental rus. Clin Infect Dis 1993; 16: 301-2. salivary glands of mice and rats. Acta Histochem Cytochem 115. Kleinnegger CL, Stoeckel DC and Kurago ZB. A compari- 1992; 25: 629-39. son of salivary calprotectin levels in subjects with and with- 100. Mori M, Yamada K, Onomura H, et al. Immunohistochemi- out oral candidiasis. Oral Surg 2001; 92: 62-7. cal localization of S100A1 and S100A6 in postnatally devel- 116. Hetland G, Talgo GJ and Fagerhol MK. Chemotaxins C5a oping salivary glands of rats. Histochem Cell Biol 1998; 110: and fMLP induce release of calprotectin (leukocyte L1 pro- 579-87. tein) from polymorphonuclear cells in vitro. J Clin Pathol 101. Mori M, Takai Y and Shrestha P. Functional role and im- Mol Pathol 1998; 51: 143-8. munohistology of novel calcium-binding S100 proteins in 117. Eversole LR, Miyasaki KT and Christensen RE. The distri- human non-neural tumors. Asahi Univ Press, Gifu. 2002; 1- bution of the antimicrobial protein, calprotectin, in normal 18. oral keratinocytes. Arch Oral Biol 1992; 37: 963-8. 102. Gnepp DR and Wick MR. Small cell carcinoma of the major 118. Eversole LR, Miyasaki KT and Christensen RE. salivary glands. An immunohistochemical study. Cancer 1990; Keratinocyte expression of calprotectin in oral inflammatory 66: 185-92. mucosal diseases. J Oral Pathol Med 1993; 22: 303-7. 103. Angeletti RH, Aardal S, Serck-Hanssen G, et al. Vasoinhibi- 119. Ross KF and Herzberg MC. Calprotectin expression by gin- tory activity of synthetic peptides from amino terminus of gival epithelial cells. Infect Immun 2001; 69: 3248-54. chromogranin A. Acta Physiol Scand 1994; 152: 1225-32. 120. Kido J, Nishikawa S, Ishida H, et al. Identification of 104. Metz-Boutigue MH, Garcia-Sablone P, Hogue-Angeletti R, calprotectin, a calcium binding leukocyte protein, in human et al. Intracellular and exracellular processing of dental calculus matrix. J Periodont Res 1997; 32: 355-61. chromogranin A: Determination of cleavage sites. Eur J 121. Kido J, Nakamura T, Kido R, et al. Carprotectin, a leukocyte Biochem 1993; 217: 247-57. protein related to inflammation, in gingival crevicular fluid. 105. Lugardon K, Raffner R, Goumon Y, et al. Antibacterial and J Priodont Res 1998; 33: 434-7. antifungal activities of vasostatin-1, the N-terminal fragment 122. Miyasaki KT, Voganatsi A, Huynh T, et al. Calprotectin and of chromogranin A. J Biol Chem 2000; 275: 10745-53. lactoferrin levels in the gingival crevicular fluid of children. 106. Sturb JM, Goumon Y, Lugardon K, et al. Antibacterial ac- J Priodontol 1998; 69: 879-83. tivity of glycosylated and phosphorylated chromogranin A- 123. Kido J, Nakamura T, Kido R, et al. Calprotectin in gingival derived peptide 173-194 from bovine adrenal medullary chro- crevicular fluid correlates with clinical and biochemical maffin granules. J Biol Chem 1996; 271: 28533-40. markers of periodontal diseases. J Clin Periodontol 1999; 107. Sturb JM, Hubert P, Nullans G, et al. Antibacterial activity 26: 653-7. of secretolytin, a chromogranin B-derived peptide. FEBS 124. Nakamura T, Kido J, Kido R, et al. The association of Letter 1996; 379: 273-8. calprotectin level in gingival crevicular fluid with gingival 108. Goumon Y, Lugard K, Kieffer B, et al. Characterization of index and the activities of collagenase and aspartate ami- antibacterial COOH-terminal proenkephalin A-derived pep- notransferase in adult periodontitis patients. J Periodontol tides (PEAP) in infectious fluid. J Biol Chem 1998; 273: 2000; 71: 631-7. 29847-56. 125. Suryono, Kido J, Hayashi N, et al. Effect of Porphyromonas 109. Johne B, Fagerhol MK, Lyberg T, et al. Functional and clini- gingivalis lipopolysaccharide, tumor necrosis factor-α, and cal aspects of myelomonocyte protein calprotectin. J Clin interleukin-1β on calprotectin release in human monocytes. Pathol Mol Pathol 1997; 50: 113-23. J Periodontol 2003; 74: 1719-24. 110. Miyasaki KT. The neutrophil: Mechanism of controlling pe- 126. Nisapakultorn K, Ross KF and Herzberg MC. Calprotectin riodontal bacteria. J Periodontol 1991; 62: 761-74. expression inhibits bacterial binding to mucosal epithelial 111. Steinbakk M, Naess-Anderesen CF, Lingaas E, et al. Anti- cells. Infect Immun 2001; 69: 3692-6. microbial actions of calcium binding leukocyte L1 protein, 127. Nisapakultorn K, Ross KF and Herzberg MC. Calprotectin calprotectin. Lancet 1990; 336: 763-5. expression in vitro by oral epithelial cells confers resistance 112. Rugtveit J, Scott H, Halstensen TS, et al. Expression of the to infection by Porphyromonas gingivalis. Infect Immun 2001; Oral Med Pathol 11 (2006) 15

69: 4242-7. 145. Sahasrabudhe KS, Kimball JR, Morton TH, et al. Expres- 128. Cuida M, Brun JG, Tynning T, et al. Calprotectin levels in sion of the antimicrobial peptide, human β-defensin 1, in oral fluid: the importance of collection site. Eur J Oral Sci duct cells of minor salivary glands and detection in saliva. J 1995; 103: 8-10. Dent Res 2000; 79: 1669-74. 129. Cuida M, Halse AK, Johannessen AC, et al. Indicators of 146. Dunsche A, Acil Y, Siebert R, et al. Expression profile of salivary gland inflammation in primary Sjögren’s syndrome. human defensins and antimicrobial proteins in oral tissues. Eur J Oral Sci 1997; 105: 228-33. J Oral Pathol Med 2001; 30: 154-8. 130. Brun JG, Cuida M, Jacobsen H, et al. Sjögren’s syndrome in 147. Zhang L, Yu W, He T, et al. Defensin antimicrobial peptides inflammatory rheumatic diseases: analysis of the leukocyte in the oral cavity. J Oral Pathol Med 2001; 30: 321-7. protein calprotectin in plasma and saliva. Scand J Rheu 1994; 148. Dunsche A, Acil Y, Dommisch H, et al. The novel human 23: 114-8. beta-defensin-3 is widely expression in oral tissues. Eur J 131. Challacombe SJ. Immunologic aspects of oral candidiasis. Oral Sci 2002; 110: 121-4. Oral Surg 1994; 78: 202-10. 149. Cole AM, Dewan P and Ganz T. Innate antimicrobial activ- 132. Greenspan D. Treatment of oral candidiasis in HIV infec- ity of nasal secretions. Infect Immun 1999; 67: 3207-15. tion. Oral Surg 1994; 78: 211-5. 150. Lee SH, Kim JE, Lim HH, et al. Antimicrobial defensin pep- 133. Isaksen B and Fagerhol MK. Calprotectin inhibits matrix tides of the human nasal mucosa. Ann Otol Rhinol Laryngol metalloproteinases by sequestration of zinc. J Clin Pathol 2002; 111: 135-41. Mol Pathol 2001; 54: 289-92. 151. Singh PK, Jia HP, Wiles K, et al. Production of β-defensins 134. Santhanagopalan V, Hahn L and Sohnle PG. Resistance of by human airway epithelia. Proc Natl Acad Sci USA 1998; zinc-supplemented Candida albicans cells to the growth in- 95: 14961-6. hibitory effect of calprotectin. J Infect Dis 1995; 171: 1389-94. 152. Diamond G, Kaiser V, Rhodes J, et al. Transcriptional regu- 135. Sohnle PG, Hahn BL and Santhanagopalan V. Inhibition of lation of β-defensin gene expression on tracheal epithelial Candida albicans growth by calprotectin in the absence of cells. Infect Immun 2000; 68: 113-9. direct contact with the organisms. J Infect Dis 1996; 174: 153. Dale BA and Krisanaprakornkit S. Defensin antimicrobial 1369-72. peptides in the oral cavity. J Oral Pathol Med 2001; 30: 321-7. 136. Chertov O, Michiel DF, Xu L, et al. Identification of defensin- 154. Zhao D, Wang I and Lehrer RI. Widespread expression of 1, defensin-2, and CAP37/Azurocidin as T-cell beta-defensin hBD-1 in human secretory glands and epithe- chemoattractant proteins released from interleukin-8-stimu- lial cells. FEBS Letters 1996; 396: 319-22. lated neutrophils. J Biol Chem 1996; 271: 2940-53. 155. Abiko Y, Nishimura M, Kusano K, et al. Presence of human 137. Ouellette AJ. Mucosal immunity and inflammation IV. beta-defensin 2 peptide in keratinization in salivary gland Paneth cell antimicrobial peptides and the biology of the tumor. Oral Med Pathol 2000; 5: 95-7. mucosal barrier. Am J Physiol 1999; 277: G257-61. 156. Bonass WA, High AS, Owen PJ, et al. Expression of β- 138. O’Neil D, Porter EM, Elewaut D, et al. Expression and regu- defensin genes by human salivary gland. Oral Microbiol lation of the human β-defensins hBD-1 and hBD-2 in intes- Immunol 1999; 14: 371-4. tinal epithelium. J Immunol 1999; 163: 6718-24. 157. Bensch KW, Raida M, Magert HJ, et al. HBD-1: a novel β- 139. Bals R, Goldman MJ and Wilson JM. Mouse β-defensin 1 defensin from human plasma. FEBS Letters 1995; 368: 331-5. is a salt-sensitive antimicrobial peptide present in epithelia 158. Abiko Y, Mitamura J, Nishimura M, et al. Pattern of expres- of the lung and urogenital tract. Infect Immun 1998; 66: 1225- sion of beta-defensins in oral squamous cell cartinoma. Can- 32. cer Letters 1999; 143: 37-43. 140. Valore EV, Park CH, Quayle AJ, et al. Human β-defensin- 159. Abiko Y, Suraweera AK, Nishimura M. et al. Differential 1: An antimicrobial peptide of urogental tissue. J Clin Invest expression of human beta-defensin 2 in keratinized and non- 1998; 101: 1633-42. keratinized oral epithelial lesions; immunohistochemistry 141. Harder J, Bartels J, Christophers E, et al. A peptide antibi- and in situ hybridization. Virchows Arch 2001; 438: 248-53. otic from human skin. Nature 1997; 387: 881. 160. Abiko Y, Jinbu Y, Noguchi T, et al. Regulation of human β- 142. Chronnell CM, Ghali LR and Ali RS. Human beta defencin- defensin 2 peptide expression in oral lichen planus, leuko- 1 and -2 expression in human pilosebaceous unit: plakia and candidiasis. An immunohistochemical study. upregulation in acne vulgaris lesions. J Invest Dermatol 2001; Pathol Res Practis 2000; 198: 537-42. 117: 1120-5. 161. Noguchi T, Jinbu Y, Kusama M, et al. Relation of expression 143. Krisanapakornkit S, Weinberg A, Perez CN, et al. Expres- pattern of β-defensin 2 in oral lichen planus with clinico- sion of the peptide antibiotic human β-defensin 1 in cul- pathological features. J Jpn Oral Muco Membr 2004; 10: 6-10. tured gingival epithelial cells and gingival tissue. Infect 162. Krisanapakornkit S, Kimball JR, Weinberg A, et al. Induc- Immun 1998; 66: 4222-8. ible expression of human β-defensin 2 by Fusobacterium 144. Mathews M, Jia HP, Guthmiller JM, et al. Production of β- nucleatum in oral epithelial cells: Multiple signaling path- difensin antimicrobial peptides by the oral mucosa and sali- way and role of commensal bacteria in innate immunity and vary glands. Infect Immun 1999; 67: 2740-5. the epithelial barrier. Infect Immun 2000; 68: 2907-15. 16 Mori et al. Antimicrobial peptides in saliva

163. Saitoh M, Abiko Y, Shimabukuro S, et al. Correlated expres- in inflammatory salivary gland tissues. J Hiroshima Univ sion of human beta defensin-1, -2, -3 mRNAs in gingival tis- Dent 1984; 16: 205-10. sues of young children. Arch Oral Biol 2004; 49: 799-803. 180. Campbell GA, Burgdorf WHC and Everett MA. The immu- 164. Schutte BC, Mitros JP, Bartlett JA, et al. Discovery of five nohistochemical localization of lysozyme in human axillary conserved b-defensin gene clusters using a computational apocrine glands. J Invest Dermatol 1982; 79: 351-3. search strategy. Proc Natl Acad Sci 2002; 99: 2129-33. 181. Van der Oord JJ, De Wolf-Peeters C and Desmet V. Immu- 165. Krisanapakornkit S, Jotikasthira D and Dale BA. Intracel- nohistochemical lacalization of lysozyme in the nasal respi- lular calcium in signaling human α -defensin-2 expession ratory mucosa. Arch Oto-Rhino-Largngol 1982; 237: 1-5. in oral epithelial cells. J Dent Res 2003; 82: 877-82. 182. Kittas C, Aroni K, Kotsis L, et al. Distribution of lysozyme,

166. Zhang L, Yu W, He T, et al. Contribution of β-defensin 1, 2 α 1-antichymotrypsin and α 1-antitrypsin in adenocarcino- and 3 to the anti-HIV-1 activity of CD8 antiviral factor. Sci- mas of the stomach and large intestine. Virchows Arch A 1982; ence 2002; 298: 995-1000. 139: 292-6. 167. Tanida T, Okamoto T, Okamoto A, et al. Decreased expres- 183. Anna-Maija R. Lysozyme (muramidase) activity of sion of antimicrobial proteins and peptides in saliva of pa- leuckocytes and exfoliated epithelial cells in oral cavity. tients with oral candidiasis. J Oral Pathol Med 2003; 32: Scand J Dent Res 1972; 80: 422-7.

586-94. 184. Ling L and Klein MJ. Lysozyme and α 1-antitrypsin in gi- 168. Mizukawa N, Sugiyama K, Ueno T, et al. Defensin-1, an ant-cell tumor of bone and in other lesions that contain gi- antimicrobial peptide presents in saliva of patients with oral ant cells. Arch Pathol Lab Med 1986; 110: 713-8. diseases. Oral Dis 1999; 5: 139-42. 185. Aroni K, Liossi A, Fotiou G, et al. An immunohistochemical 169. Biragyn A, Ruffini PA, Leifer CA, et al. Toll-like receptor 4- study of normal human neonate and adult parotid gland tis-

dependent activation of dendritic cells by β-defensin 2. Sci- sue. Detection of lysozyme, lactoferrin, α1-antichymotrypsin,

ence 2002; 298: 1025-9. α1-antitrypsin and carcinoembryonic antigen. Path Res Pract 170. Fionnuala T, Lundy FT, Orr DF, et al. Identification and 1988; 183: 292-6. overexpression of human neutrophil α -defensins (human 186. Lee SK, Lim CY, Chi JG, et al. Immunohistochemical local-

neutrophil peptides a, 2 and 3) in aquamous cell cartinoma ization of lysozyme, lactoferrin, α 1-antichymotrypsin and

of the human toung. Oral Oncol 2004; 40: 139-44. α 1-antitrypsin in salivary gland of human fetus. Acta 171. Mizukawa N, Sugiyama K, Kamino M, et al. Immunohis- Histochem 1990; 89: 201-11. tochemical staining of human alpha defensin 1 (HNP-1), in 187. Tourville DR and Adler RH. The human secretory immuno- the submandibular glands of patients with oral carcinoma. globulin system: Immunohistological lacalization of γA, Anticancer Res 2000; 20: 1125-7. secretory “piece”, and lactoferrin in normal human tissues. 172. Brogden KA, Heidari M, Sacco RE, et al. Defensin-induced J Exp Med 1969; 129: 411-29. adaptive immunity in mice and its potential in preventing 188. Tabak L and Mandel, ID. Alterations in lactoferrin in sali- periodontal disease. Oral Microbiol Immunol 2003; 18: 95-9. vary gland disease. J Dent Res 1978; 57: 43-7. 173. Bissell J, Joly S, Johnson GK, et al. Expression of β- 189. Reitamo S, Konttinen YT and Raeste AM. Distribution of defensins in gingival health and in periodontal disease. J lactoferrin in human salivary glands. Histochemistry 1980; Oral Pathol Med 2004; 33: 378-85. 66: 285-91. 174. Mori M. Histochemistry of the salivary glands. CPC Press, 190. Shested M, Barfoed C, Krogdahl A, et al. Immunohistochemi-

Boca Raton. 1991; 109-88. cal investigation of lysozyme, lactoferrin, α 1-antitrypsin,

175. Reitamo S, Klockars M and Raeste AM. Immunohistochemi- α 1-antichymotrypsin and ferritin in parotid gland tumors. cal identification of lysozyme in the minor salivary glands of J Oral Pathol 1985; 14: 459-63. man. Arch Oral Biol 1977; 22: 515-9. 191. Tsukitani K, Nakai M, Tatemoto Y, et al. Histochemical stud- 176. Caselitz J, Jaup T and Seifert G. Lactoferrin and lysozyme ies of obstructive adenoma in human submandibular sali- in cartinomas of the parotid gland. Virchows Arch A 1981; vary glands. Immunohistochemical demonstration of 394: 61-73. lactoferrin, lysozyme and carcinoembyonic antigen. J Oral 177. Korsrud FR and Brandtzaeg P. Characterization of epithe- Pathol 1985; 14: 631-8. lial elementsin human major salivary glands by functional 192. Mitani H, Murase N and Mori M. Immunohistochemical markers; Locarization of amylase, lactoferrin, lysozyme secre- demonstration of lysozyme and lactoferrin in salivary pleo- tory component, and secretory immunoglobulins by paired morphic adenomas. Virchow Arch B 1989; 57: 257-65. immunofluorescense staining. J Histochem Cytochem 1982; 193. Chomette G, Auriol M, Vaillant JM, et al. An immunohis- 30: 657-66. tochemical study of the distribution of lysozyme, lactoferrin,

178. Moro I, Umemura S, Crago SS, et al. Immunohistochemical α1-antitrypsin and α1-antichymotrypsin in salivary adenoid distribution of immunoglobulines, lactoferrin, and lysozyme in cystic carcinoma. Pathol Res Prac 1991; 187: 1001-8. human minor salivary glands. J Oral Pathol 1984; 13: 97-104. 194. Gauldie J, Lamontagne L, Horsewood P, et al. Immunohis-

179. Ogawa I, Takata T, Ogura M, et al. Immunohistochemical tochemical localization of α 1-antitrypsin in normal mouse lacalization of lactoferrin, lysozyme and secretory component liver and pancreas. Am J Pathol 1980; 101:723-35. Oral Med Pathol 11 (2006) 17

195. Papadimitriou CS, Stein H and Papacharalampous NX. 82: 944-50.

Presence of α 1-antichymotrypsin and α 1-antitrypsin in 212. Nikawa H, Fukushima H, Makihira S, et al. Fungicidal ef- haematopoietic and lymphoid tissue cells as revealed by the fect of three new synthetic peptides against Candida immunoperoxidase method. Path Pes Pract 1980; 169: 287-97. albicans. Oral Dis 2004; 10: 221-8. 196. Geboes K, Ray MB, Rutgeerts P, et al. Morphological identi- 213. Huang CM. Comparative proteomic analysis of human whole fication of alpha-1-antitrypsin in human small intestine. saliva. Arch Oral Biol 2004; 49: 951-62. Histopathology 1982; 6: 55-60. 214. Matsuzaki K. Why and how are peptide-lipid interactions 197. Kittas C, Aroni K, Matani A, et al. Immunohistochemical utilized for self-defense? and tachyplesins as ar-

demonstration of α 1-antitrypsin and α 1-antichymotrypsin chetypes. Biochim Biophys Acta 1999; 1462: 1-10 in human gastrointestinal tract. Hepatogastronterol 1982; 215. Epand RM and Vogel HJ. Diversity of antimicrobial pep- 29: 275-7. tides and their mechanisms of action. Biochim Biophys Acta 198. Ordonez NG, Manning JT and Hanssen G. Alpha1-anti- 1999; 1462: 11-28. trypsin in islet cell tumors of the pancreas. Am J Clin Pathol 216. Sitaram N and Nagaraj R. Interaction of antimicrobial pep- 1983; 80: 277-82. tides with biological and model membrane: structural and 199. Tahara E, Ito H, Taniyama K, et al. Alpha-antitrypsin, al- charge requirements for activity. Biochim Biophys Acta 1999; pha-antichymotrypsin, and alpha 2-macroglobulin in human 1462: 29-54. gastric carcinomas: A retrospective immunohistochemical 217. O’Connell BC, Zheng C, Jacobson-Kram D, et al. Distribu- study. Hum Pathol 1984; 15: 957-64. tion and toxicity resulting from adenoviral vector adminis- 200. Nakhleh RE and Snover DC. Use of alpha1-antitrypsin stain- tration to a single salivary gland in adult rats. J Oral Pathol ing in the diagnosis of nodular regenerative hyperplasia of Med 2003; 32: 414-21. the liver. Hum Pathol 1988; 19: 1048-52. 218. Kikuchi R, Watanabe N, Konno T, et al. High incidence of 201. Soini Y and Miettinen M. Widespread immunoreactivity for silent aspiration in elderly patients with community-acquired alpha1-antichymotrypsin in different types of tumors. Am J pneumonia. Am J Crit Care Med 1994; 150: 251-3 Clin Pathol 1988; 90: 131-6. 219. Limeback H. Implication of oral infections on systemic dis- 202. Maruse N, Kobayashi K, Mitani H, et al. Immunohistochemi- eases in the institutionalized elderly with a specific focus on

cal localization of α1-antitrypsin and α1-antichymotrypsin pneumonia. Ann Periodontol 1998; 3: 262-75. in salivary pleomorphic adenomas. Virchows Arch A 1985; 220. Abe S, Ishikawa I and Okuda K. Prevalence of potential 408: 107-16. respiratory pathogens in the mouths of elderly patients and 203. Weinberg ED. Human lactoferrin: a novel therapeutic with effect of professional oral care. Arch Gerontol Geriat 2001; broad spectrum potential. J Pharm Pharmacol 2001; 53: 32: 45-55. 1303-10. 221. Lee SK. Mucocidin, A novel antimicrobial peptide gene ex- 204. Tenovuo J. Clinical applications of antimicrobial host pro- pressed in the human mucosal epithelial cells and exocrine teins lactoperoxidase, lysozyme and lactoferrin in xerosto- glands. Int Symposium Maxillo-oral Reg Biol in OKAYAMA mia efficacy and safety. Oral Dis 2002; 8: 23-9. 2005; Programe and Abstract: 231. 205. Soares RV, Siqueira CC, Bruno LS, et al. MG2 and lactoferrin from a heterotypic complex in salivary secretions. J Dent (Accepted for publication March 25, 2006) Res 2003; 82: 471-5. 206. Soares RV, Lin T, Siqueira CC, et al. Salivary micelles: iden- tification of complexes containing MG2, sIgA, lactoferrin, amylase, glycosylated proline-rich protein and lysozyme. Arch Oral Biol 2004; 49: 337-43. 207. Tenovuo J, Moldoveanu Z, Mestechy J, et al. Interaction of specific and innate factors of immunity: IgA enhances the antimicrobial effect of the lactoperoxidase system against Streptococcus mutans. J Immunol 1982; 128: 726-31. 208. Crawford JM, Taubman M and Smith DJ. Minor salivary glands as a major source of secretory immunoglobulin A in the human oral cavity. Science 1975; 190: 1206-8. 209. Smith DJ, Taubman MA and All-Salaam P. Immunoglobu- lin isotypes in human minor salivary gland saliva. J Dent Res 1991; 70: 167-70. 210. Nieuw Amerongen AV and Veerman ECI. Saliva - the de- fender of the oral cavity. Oral Dis 2002; 8: 12-22. 211. LeClair EE. Four reasons to consider a novel class of innate immune molecules in the oral epithelium. J Dent Res 2003;