Reactive Oxygen and Oral Defense System 9 surfaces. Saliva also contains several specific and nonspe- cific defense factors. Salivary IgA is the principal specific defense factor of saliva. The nonspecific defense factors in- clude mucins, salivary glycoproteins, lactoferrin, lysozyme, peroxidase, histatins, and cystatins. The mucins form a vis- cous slime layer on oral mucosa that traps microorganisms and antigens, limiting their penetration into the tissues [1, 2]. Potentially harmful microorganisms are thus eliminated by the continuous renewal of the mucous layer combined with the washing action of salivary flow. Lysozyme can lyse some bacterial species by hydrolyzing glycosidic linkages in the cell wall peptidoglycan. It may also cause lysis of bacte- rial cells by interacting with monovalent anions, such as thiocyanate, perchlorate, iodide, bromide, bicarbonate, ni- trate, and fluoride, and with proteases found in saliva. Sali- vary peroxidase is produced by salivary gland acinar cells and salivary myeloperoxidase is released from oral neutro- phils. These enzymes are part of an antimicrobial system that involves the oxidation of salivary thiocyanate to hy- pothiocyanite and hypothiocyanous acid by hydrogen per- oxide (1), generated by oral bacteria and neutrophils. It is known that thiocyanite anion has the strong cell toxicity. It is thought that this compound plays a major role to the host de- fense in oral cavity. In addition, the oxidizing agents react with sulfhydryl groups of the enzymes (2 and 3) involved in glycolysis and sugar transport [3]. Then, these enzymes were inactivated. H2O2 + SCN− + H+ → HOSCN + H2O ......................... (1) RSH + OSCN− → RS-SCN + OH−............................... (2) RS-SCN + H2O → RSOH + SCN− + H+ ...................... (3) The gingival crevice of the oral cavity is almost essen- tially controlled by the antimicrobial factors of plasma. Cel- lular and humoral components of blood can reach the gin- gival crevice. Even in the healthy state, there is a continuous flow of small quantities of fluid and leukocytes from the gin- gival capillaries through the crevicular epithelium into the gingival crevice. This flow increases markedly with inflam- mation. The flow of gingival fluid from the crevice to the oral cavity removes nonadherent bacteria. Gingival fluid also contains antimicrobial substances including IgM, IgG, IgA, complement, and leukocytes. These factors are prima- rily protective against microbial invasion. Complement fac- tors may initiate bacterial cell lysis or enhance phagocytosis of microorganisms. The IgG antibodies may enhance phago- cytosis and killing of oral microorganisms through activa- tion of complement or opsonization [4–6]. Lysozyme and peroxidase that are released from the lysosome of neutro- phils during phagocytosis might also control microbial growth in the gingival crevice. Vol. 42, No. 1, 2008 Production of Superoxide and Nitric Oxide (NO) in Oral Cavity and Defense System Oral neutrophils Neutrophils play crucial roles in protecting hosts against invading microbes and in the pathogenesis of inflammatory tissue injury. Although neutrophils migrate into mucosal layers of digestive and respiratory tracts, only limited infor- mation is available of their fate and function in situ. We pre- viously reported that, unlike circulating neutrophils, neutro- phils in the oral cavity spontaneously generate superoxide radical and nitric oxide in the absence of any stimuli. Large numbers of neutrophils migrate through the mucosal layers of the intestinal and respiratory tracts and appear in their lu- minal compartments. The leukocytes in oral cavity are com- posed of 90% polymorphonuclear leukocytes (PMNs, neu- trophils) and 10% mononuclear cells. About 80% of neutro- phils are viable and functional within the oral cavity and crevice. The circulating neutrophils have been known to be primed by various ligands, such as lipopolysaccharide (LPS), interleukin-1, and TNF-α [7, 8]. The circulating neu- trophils undergo priming during the migration into the oral cavity and spontaneously release reactive oxygen species, including superoxide, hypochloride, and nitric oxide [9]. Oral neutrophils are further activated by various ligands. It was realized that components of Gram-negative bacterial cell walls, such as LPS, are able to enhance the oxidative burst of human circulating neutrophils in response to sec- ondary stimulation without themselves initiating any mea- surable activity of the NADPH oxidase [10–12]. Large num- bers gram-negative appear in oral cavity. Gram-negative or- ganisms contain LPS in their outer membranes, and this may be released during infection. It has been demonstrated that LPS elicits TNF-α production [13]. It is well known that the priming of circulating neutrophils by LPS and TNF-α results in an enhanced oxidative burst and enhanced degranulation [7, 8, 14]. Since Gram-negative microorganisms predomi- nate in the subgingival environment, and bacteremia and en- dotoxemia are commonly observed even during normal oral hygiene procedures, oral neutrophils always primed by these factors. Fate of oral neutrophils The life span of neutrophils is fairly short compared with those of other leukocytes [15]. Even under physiological conditions, neutrophils spontaneously undergo apoptosis in the absence of any stimuli, and apoptotic cells are phagocy- tosed by macrophages [16–18]. The life cycle of neutrophils is regulated by many factors, including granulocyte mac- rophage colony-stimulating factor (GM-CSF) [19, 20], nerve growth factor [21], interleukin-1 [22], interleukin-2 [23], and granulocyte colony-stimulating factor (G-CSF) [20, 24]. Unlike circulating neutrophils, oral neutrophils