Saliva – the defender of the oral cavity

It is generally accepted that saliva is of paramount importance for the maintenance of oral health. This is based on the numerous studies reporting subjective and objective functional losses that occur in persons who lack the ability to produce adequate volumes of saliva. These include dry mouth feeling (xerostomia), difficulty with swallowing food, and an increased susceptibility for opportunistic infections. The last issue points to an active protective role of saliva in maintaining oral health under normal conditions. The mild climate present in the oral cavity, i.e. an elevated temperature, a high humidity and regular supply of foodstuffs, fosters the growth of a myriad of different aerobic and anaerobic microorganisms, which together form a complex and stable ecosystem. For example, the oral mucosal surfaces of the newborn infant are the portal of entry for the majority of pathogenic microorganisms from the first day of life (92). Saliva plays a key role in maintaining the steady-state of this system, as becomes clear when the salivary clearing is blocked, for instance in sedated patients in intensive care. In the majority of these patients within 2 weeks a shift in the oral microflora occurs to Gram-negative species, which subsequently spread into the respiratory tract causing pulmonary afflictions. This is one example of the crucial role played by saliva in the maintenance of general health, but similar observations can be made in other patients suffering from an impaired saliva secretion. It is recognized for years that saliva contains many components that, in one way or another interact with microorganisms, in this way controlling the composition of the oral microflora. In the seventies and eighties of the previous century, the main proteins and peptides in human saliva have been identified and characterized (see Figure 1). Still for a lot of proteins the precise biological role remained elusive as translation of biochemical properties to biological functions proved to be difficult or resulted in erroneous concepts. In the seventies, research focused on elucidation of the role played by saliva in the protection of dental enamel and identified a large number of proteins that in vitro were involved in the formation of pellicles on hydroxyapatite. Henceforth they were attributed a role in the protection of tooth surfaces. The insight that a lot of so-called saliva-specific or pellicle-specific proteins also were present in other parts of the human body has stimulated further investigation to the biological role in the innate protection of mucous oral epithelia (90). As a consequence, for some salivary proteins the existing concepts were refined, while for others a completely different role was found, e.g. as microbicidal agents, or as physiological inhibitors of proteinases. Schematic presentation of the main functions of saliva in relation to its constituents In earlier days the immunoglobulins in saliva have received much attention in relation to their specific protective function to a single type of microorganism. However, nowadays it has become more clear that, in addition to this acquired immune system, also an innate immune system has been secreted into saliva. In the last years more light has been shed on the protective functions of the peptides and of the (glyco)proteins of the innate immune system, contributing to the first line of oral defence (27). In addition, recent research on bacteriostatic (glyco)proteins revealed that they have hidden domains possessing microbicidic properties that come available after proteolysis. Although it appears at first glance on Figure 1 that a redundance to defence mechanisms in saliva is present (85), this has been suggested only by in vitro studies. In vivo it is clear that the inhibiting and killing effects are regulated precisely so that an ecology exists in an equilibrium system in the oral cavity. This mini-review, primarily dealing with the defensive systems in saliva, will in particular focus on the more recently obtained data, aiming to (re)interpret earlier observations on the basis of our present knowledge. In this respect it is intended to give an update of the current knowledge and insights in the protective role of salivary components. The earliest studies on salivary mucins concluded that these glycoproteins occurred as a single high molecular weight species containing blood group activity (68). Later studies, using more stringent isolation and analytical techniques demonstrated that human saliva contains two genetically distinct mucin types, designated MG1 and MG2 (56; 76; 100), originating from the MUC5B and MUC7 gene, respectively (12; 22). MUC7 (MG2) has been found to exist as at least two glycoforms, MG2a and MG2b, respectively (8). In addition, genetic polymorphism has been shown for MUC7, which is associated with asthma (50). Salivary MUC5B (MG1), which displays blood group activity, exists in at least three different glycoforms, differing in sialic acid and sulphate content, depending on the glandular source (112; 14). Even within one glandular secretion, different MUC5B glycoforms could be distinguished (112, 115; 14; 103), highlighting the extreme inter- and intramolecular heterogeneity of this class of salivary glycoproteins. MUC5B functionally and structurally belongs to the classical mucins, which are the main constituents of the slime layers that cover the mucous epithelia throughout the body, e.g. in the gastrointestinal tract, the urogenitary tracts, and the respiratory tracts. These secretory mucins, although encompassing a genetically heterogeneous family, have a comparable architecture: they are composed of disulphide linked monomers that contain heavily glycosylated domains, interspersed with less glycosylated peptide domains (`naked' peptides). Because of their high carbohydrate content (>80%), their large dimensions (>1 μm), and their extended thread-like structure, the classical secretory mucins already at low concentrations form hydrophilic viscoelastic gels. These gels function as barriers, protecting the underlying epithelium against mechanical damage and preventing direct entrance of noxious agents, including bacteria and viruses, into the underlying vulnerable epithelium. In the oral cavity, MUC5B is present in the protein films (pellicles) covering the enamel and epithelial surfaces, and in this quality protect against acidic attacks (75) and modulate the microbial colonization of these surfaces (Table 1). Besides in the mucous acini of the (sero)mucous salivary glands (submandibular, sublingual, palatal and labial glands), MUC5B is also expressed in other body tissues, including the submucosal tracheobronchial glands, the gall bladder and the endocervix (30; 93; 109). In contrast, the expression of MUC7 is confined largely to the serous cells of the (sero)mucous salivary glands and of the respiratory tracts (8; 74; 93; 13). MUC7 and MUC5B are structurally and functionally completely different molecules. MUC7 is a relatively small (Mr 125 kDa) monomeric species, having low viscoelastic properties. The carbohydrate side chains of MUC7 are predominantly sialylated di- and trisaccharides, while those in MUC5B are much more heterogeneous, with sizes that may vary between two and >20 residues. Furthermore, in contrast to MUC5B, MUC7 lacks blood group active carbohydrates of the ABO-system, a feature that was considered a characteristic trait of mucins (80). When in vitro tested, preparations containing high molecular weight blood group reactive glycoproteins cause a wide variety of oral microorganisms to agglutinate, highlighting binding of these components with bacterial receptors. Extrapolated to in vivo situations, it is tempting to speculate that this is a physiological mechanism for clearing, to diminish overload of bacteria. It has become clear now that MUC7 and the salivary agglutinin (see below) are among the major bacteria-agglutinating factors in saliva. Several studies have reported binding of MUC7 to a variety of microorganisms, e.g. Streptococcus sanguis, S. mitis, S. gordonii, Actinobacillus actinomycetemcomitans, Pseudomonas aeruginosa and Escherichia coli (72; 83; 31; 70). In addition to carbohydrate residues, e.g. sialic acid (83; 31), unglycosylated peptide domains in MUC7 are involved in the interaction with bacteria (70; 59; 33). Interestingly, the histidin-rich N-terminal peptide domain of MUC7, which encompasses residues 23–37, by itself has bactericidal properties, probably because it is able to bind and disturb the bacterial membranes (60; 96). Studies aimed at identification of species specifically binding to (isolated) MUC5B preparations have demonstrated only few species capable of binding to this mucin, including Haemophilus parainfluenzae and Helicobacter pylori (113, 114; 15), whereas other species, including Streptococci do not bind (72). Salivary agglutinin is a highly glycosylated protein, with a molecular mass of approximately 340 kDa, that carries blood group active antigens. Except for the presence of blood group antigens, salivary agglutinin, which is identical to gp-340 expressed in lung (45; 81; 58) shares a number of features with MUC7: both are monomeric, heavily glycosylated proteins, with extremely sticky properties. As a consequence, under native conditions these proteins occur associated with a variety of salivary proteins, including S-IgA (7; 66). Both MUC7 and salivary agglutinin are expressed in the serous cells of the submandibular, sublingual and labial glands (93; 9). However, agglutinin, in contrast to MUC7, is also synthesized in the serous parotid gland. In the parotid gland agglutinin was localized only in the ductal cells, whereas in the submandibular gland agglutin was detected in both serous acinar cells and serous demilune cells capping the mucous acini (9). Agglutinin, initially identified as the protein responsible for the S. mutans aggregating properties of parotid saliva (26), was later found to mediate also the binding between S. mutans and S. sanguis (54). This salivary glycoprotein, which very likely is identical to the Eikenella aggregating factor (EcAF) isolated by 24), is also detectable as a component of the salivary pellicle on the tooth surface (19). It now has become clear that agglutinin, like MUC7, binds to a wide variety of microorganisms, including S. mutans, S. salivarius and S. sanguis (57). Evidence has been produced that salivary agglutinin is genetically very similar, if not identical, to gp-340, a glycoprotein present in lung washings (81; 58). Gp-340 binds to surfactant protein-D, resulting in enhanced phagocytosis and killing of microorganisms by neutrophils and macrophages (45). Gp-340 is also identical to DMBT-1 (protein Deleted in Malignant Brain Tumours), a member of the scavenger receptor family, a group of proteins that, because of their capacity to bind to a broad variety of ligands, has been implicated in the innate immune system (69). In the eighties it became apparent that a group of human salivary proteins, which initially were implicated in the control of mineralization, possesses cysteine proteinase inhibitory activity (94). 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