ORIGINAL PAPERS
Anna Zalewska
1, A, D, E, F,, Małgorzata Knaś
2, B, C, F, Julita Szulimowska
3, B, F,
Napoleon Waszkiewicz
4, C, D, Katarzyna Wołosik
2, D, E, Danuta Waszkiel
1, A, DNonspecific Immune Factors
in the Whole Unstimulated Saliva
of Human Infants, Children and Adolescents*
Białka odporności nieswoistej w ślinie całkowitej niestymulowanej
niemowląt, dzieci i młodzieży
1 Department of Conservative Dentistry Medical University, Białystok, Poland 2 Insitute of Health Care, Higher Vocational School, Suwałki, Poland
3 Department of Pedodontics Medical University, Białystok, Poland
4 Department of Psychiatry Medical University, Białystok, Choroszcz, Poland
A – concept, B – data collection, C – statistics, D – data interpretation, E – writing/editing the text, F – compiling the bibliography
Abstract
Background. The first and the earliest actuated line of defense against pathogens are non-specific defense
mecha-nisms of the innate immune response. Non-specific (innate) immune resistance depends on the destruction of pathogens by specific proteins and phagocytic cells.
Objectives. The aim of our work was the evaluation of parameters responsible for the innate immunity of saliva in
infants, children and adolescents.
Material and Methods. Enrolled in the study were 80 children and adolescents, aged from 3 months to 18 years.
Participants were divided into four study groups: toothless, group of deciduous, mixed and permanent teeth. The unstimulated salivary flow, the specific activity of peroxidase (colorimetry), the total amount of lysozyme (radial immunodiffusion) and lactoferrin (ELISA) were determined.
Results. The median value of the unstimulated flow rate in the deciduous group was significantly lower in
com-parison to the mixed and permanent groups. The specific activity of peroxidase in the saliva: of toothless group was significantly lower in comparison to all remaining groups; of the deciduous group was significantly lower in comparison to the mixed and permanent groups. A significantly higher value of the total lysozyme was observed in mixed and permanent groups in comparison to the toothless and deciduous groups. The total lactoferrin in the saliva of the toothless group was significantly higher in comparison to the deciduous group. The median value of lactoferrin in the saliva: of the deciduous group was significantly lower in comparison to the mixed and permanent groups; of the mixed group was significantly lower in comparison to the permanent group.
Conclusions. The quality of innate immunity in the saliva of children depends on the age of the child. The changes
observed in the present study in the total value of lactoferrin and lysozyme and in the specific activity of peroxidase in the saliva may have implications for bacterial colonization in the oral cavity of children and adolescents (Dent.
Med. Probl. 2013, 50, 3, 291–297).
Key words: children, lactoferrin, lysozyme, peroxidase, saliva.
Streszczenie
Wprowadzenie. Pierwszą i najszybciej uruchamianą linią obrony przed patogenami są nieswoiste mechanizmy
obronne wrodzonej odpowiedzi immunologicznej. Niespecyficzna (wrodzona) odporność immunologiczna zależy od zniszczenia czynników chorobotwórczych z udziałem specyficznych białek i komórek fagocytujących.
Dent. Med. Probl. 2013, 50, 3, 291–297
ISSN 1644-387X © Copyright by Wroclaw Medical University and Polish Dental Society
The first and at earliest actuated line of defense against pathogens are non-specific defense mech-anisms of the innate immune response. Non-spe-cific (innate) immune resistance depends on the destruction of pathogens by specific proteins and phagocytic cells. A newborn human organism has learned only the beginning of the immune system, so-called nonspecific immunity. With the growth and development of the organism, the quality of resistance also changes. Acquired resistance (spe-cific) is formed. Saliva contains a number of non-specific defense mechanisms to preserve the eco-logical balance of the oral cavity. The purpose of the defense is to reduce salivary bacterial coloniza-tion on teeth and mucous membranes and to pre-vent the penetration of harmful substances into the body. The innate immune system of the oral cavity includes e.g.: peroxidase, lysozyme and lac-toferrin.
Salivary peroxidase is the main salivary anti-oxidative enzyme. The products of peroxidase ac-tivity reveal strong antibacterial actions [1–3]. Per-oxidase activity is associated mainly with perox-idase secreted by acinar cells of the parotid and submandibular glands [2, 4]. The antimicrobial activity of lysozyme is linked with its lytic action on bacteria by catalyzing the hydrolysis of the cell wall polysaccharides. Lysozyme is also known to activate bacterial autolysins [5–7]. Lactoferrin is an iron-binding protein and deprives bacteria of this indispensable element and thus inhibits bac-terial metabolism [8]. In an iron-independent way, lactoferrin damages the outer membranes of sali-vary bacteria and generates nitric oxide in macro-phages, which kills the microorganisms [9]. The source of salivary lactoferrin and lysozyme is the ductal cells of the salivary glands; they are also derived from neutrophils and macrophages in re-sponse to oral infection [10, 11].
In the literature, we did not find data on changes in the total amount of protein responsi-ble for innate immunity of saliva depending on the age of child. Therefore, the aim of our work was to evaluate the parameters responsible for the in-nate immunity of saliva in infants, children and adolescents.
Material and Methods
Enrolled in the study were 80 children and adolescents, aged from 3 months to 18 years from the Pedodontics Department Medical University in Bialystok. Children and adolescents with any oral lesions (oral mucositis, oral candidiasis) as well as with severe gingival inflammation (change in shape, color and spontaneous gingival bleeding – also in a site of a tooth eruption) and active car-ies lesions were excluded from the study. Children with a medical history of present or previous re-current infections, with a history of asthma, al-lergy, atopic diseases, any suspected immunolog-ical disorder or gluten enterophaty were exclud-ed from the study. The children who participatexclud-ed in the study were considered healthy. Participants were divided into 4 study groups:
NT – toothless – 20 children (mean age 4 ± 1 mon-ths, 12 girls, 8 boys), all children have been breast-fed,
D – group of deciduous teeth – 20 children with all deciduous teeth present, (mean age 5 ± 2 years, 10 girls, 10 boys), none of the children have be-en breast-fed during the last two months pre-ceding the saliva collection,
M – group of mixed teeth – 20 children (mean age 9 ± 2 years, 10 girls, 10 boys),
P – group of permanent teeth – 20 adolescents (mean age 15 ± 3 years, 10 girls, 10 boys).
Cel pracy. Ocena parametrów wrodzonej odporności w ślinie niemowląt, dzieci i młodzieży.
Materiał i metody. Do badania zakwalifikowano 80 dzieci i młodzieży w wieku od 3 miesięcy do 18 lat. Uczestnicy
zostali podzieleni na 4 grupy: nieuzębioną, z uzębieniem mlecznym, mieszanym i stałym. Oznaczano wydzielanie śliny niestymulowanej, aktywność specyficzną peroksydazy (metodą kolorymetryczną), całkowitą zawartość lizozy-mu (metodą imlizozy-munodyfuzji radialnej) i laktoferyny (metodą ELISA).
Wyniki. Wydzielanie śliny niestymulowanej w grupie dzieci z uzębieniem mlecznym było istotnie mniejsze
w porównaniu z grupą dzieci z uzębieniem mieszanym i stałym. Aktywność specyficzna peroksydazy w ślinie: dzieci nieuzębionych była istotnie mniejsza w porównaniu z wszystkimi pozostałymi grupami i w grupie dzieci z uzębieniem mlecznym była istotnie mniejsza w porównaniu z grupami dzieci z uzębieniem mieszanym i stałym. Istotnie większą całkowitą zawartość lizozymu obserwowano w grupach dzieci z uzębieniem mieszanym i stałym w stosunku do dzieci bezzębnych i dzieci z uzębieniem mlecznym. Całkowita zawartość laktoferyny w ślinie dzieci bezzębnych była istotnie większa w porównaniu z dziećmi z uzębieniem mlecznym. Całkowita zawartość laktofery-ny w ślinie: dzieci z uzębieniem mleczlaktofery-nym była istotnie mniejsza w porównaniu z dziećmi z uzębieniem mieszalaktofery-nym i stałym, u dzieci z uzębieniem mieszanym była istotnie mniejsza w porównaniu z dziećmi z uzębieniem stałym.
Wnioski. Jakość odporności wrodzonej w ślinie dzieci zależy od wieku badanych. Obserwowane zmiany całkowitej
zawartości laktoferyny i lizozymu oraz aktywności specyficznej peroksydazy w ślinie mogą wpływać na rodzaj kolo-nizacji bakteryjnej jamy ustnej dzieci i młodzieży (Dent. Med. Probl. 2013, 50, 3, 291–297).
Clinical dental and gingival status, oral hy-giene examinations were performed by one pedo-dontist (J.Sz.) under the standardized conditions in the dental office using a dental mirror, probe and periodontal probe.
The status of hard tissue of the teeth was de-termined using DMFT (M, P groups) and dft (D, M groups). We decided not to assess m index in chil-dren with mixed and deciduous dentition because of the possibility of physiological loss of deciduous teeth [12]. The gingival status was examined (in D, M, P groups) using the gingival index GI, oral hygiene was determined (in D, M, P groups) using aproxi-mal plaque index (API) (Table 1). In 20 patients, the inter-rater agreements between the examiner (J.Sz.) and another experienced dentist (A.Z.) were as-sessed. The reliability for: DMFT was r = 0.98; dft was 0.99, GI was r = 0.96; API was r = 0.94.
Written informed consent was obtained from each patient and/or his/her parents, after the aims and methodology of the study were explained. The study was approved by the Ethics Committee of the Medical University of Bialystok, Poland (R-I- -002/329/2011).
Unstimulated
Saliva Collection
Children were instructed to refrain from food and beverages, except water, for one hour before having their saliva collected. All salivary sam-ples were collected between 9 a.m. and 11 a.m. be-fore the clinical examination. Saliva was collect-ed in a plastic tube placcollect-ed on ice, using the spit-ting method, under standardized conditions for 10 min [13]. With toothless children the whole saliva sample was collected from the floor of the mouth by the aspiration technique using a plastic pipette. During the collection the children were
not crying and were in the sitting position. No stimulation was used. The volume of each sam-ple (in D, M, P groups; it was impractical to de-termine reliably the flow rate of the saliva in hu-man infants (NT group)) was measured by a pi-pette calibrated in 0.1 ml units. A salivary flow rate was calculated from salivary volume, divided by the time needed for salivary sample collection. Salivary samples were centrifuged at 3,000 × g for 20 min at 4°C, to remove cells and debris. The ob-tained supernatants were divided into 200 µl por-tions, frozen and kept at –80°C, until analyzed with the exception of saliva portions for peroxi-dase activity, which was determined immediately after centrifugation. All of the saliva was collected between January and May 2011.
The sialochemical analysis included: perox-idase activity (Px), lysozyme (Lz) and lactoferrin (Lf) concentrations. The salivary flow from NT group was not actively stimulated but the children constantly moved their tongues and lips, as well as bit the pipette which could have caused some mechanical stimulation of saliva flow. To com-pensate for any stimulation of secretion rate that might have occurred during sampling we calculate Lz and Lf contents as a total value (ratio of specific protein to total protein) and Px as the specific ac-tivity (ratio of enzyme to total protein).
Activity of peroxidase was determined color-imetrically according to Mansson-Rahemtulla et al. [14]. Salivary lysozyme concentration was deter-mined by radial immunodiffusion (Human NL Na-norid plate, GT073.3, The Binding Site, Birming-ham, UK) as described Mancini, Carbonara and Heremans [15]. The concentration of lactoferrin was determined by ELISA (Bioxytech Lactof EIA – Oxis Health Products, 21015, Inc., Burlingame, CA, USA). Protein content was determined by bicincho-ninic acid BCA method (PIERCE BCA Protein As-say Kit). All analyses were performed in duplicate.
Table 1. Median values of examined stomatological parameters Tabela 1. Mediana wartości badanych parametrów stomatologicznych
API (%) GI DMFT/dft
median min max p median min max p median min max p D 40.0 35 45 D:M M:P D:P – ns 0.51 0.25 0.51 D:M M:P D: P – ns /5.6 /4.9 /6.5 Ddft:Mdft MDMFT: PDMFT P – ns M 40.8 30 47 0.54 0.2 0.6 8.9/5.3 7.0/4.0 9.0/6.0 P 42.0 25 45 0.87 0.31 0.9 10.6/ 9.2/ 11.0/
API – approximal plaque index; GI – gingival index; DMFT – number of decayed, missing and filled permanent teeth, dft – number of decayed and filled deciduous teeth; D – children with deciduous teeth, M – children with mixed dentition, P – children with permanent dentition, ns – no significant differences between groups, p < 0.05 (Kruskall-Wallis test). API – aproksymalny wskaźnik płytki; GI – wskaźnik dziąsłowy; PUW – liczba zębów stałych z próchnicą, usuniętych i wypełnionych z powodu próchnicy; pw – liczba zębów mlecznych z próchnicą i wypełnionych z powodu próchnicy; D – grupa dzieci z uzębieniem mlecznym, M – grupa dzieci z uzębieniem mieszanym, P – grupa dzieci z uzębieniem stałym, ns – brak różnic statystycznych między grupami, p < 0,05 (test Kruskala-Wallisa).
Statistical analysis was performed using Statis-tica 10.0 (software). As all the parameters did not have a normal distribution, Kruskal-Wallis ANO-VA and median test were used to study the signifi-cant differences between groups. Spearman’s rank correlation coefficient was used to study the asso-ciations between the variables. The statistical sig-nificance was defined as p ≤ 0.05.
Results
Specific Activity
of Peroxidase (Px)
The median value of the specific activity of Px in the unstimulated saliva of NT group was signif-icantly lower in comparison to D, M and P groups (p = 0.02, p < 0.0001 and p < 0.0001 respectively). Similarly, the median value of the specific activ-ity of Px in the unstimulated saliva of D group was significantly lower in comparison to M and P groups (p = 0.03 and p = 0.02 respectively). The median of the specific activity of Px in the unstim-ulated saliva of M and P groups were not signifi-cantly different from one another (Fig. 1A).
Lysozyme
A significantly higher value of the total lyso-zyme was observed in M and P groups in compar-ison to NT (p < 0.0001 and p < 0.0001 respectively) and D groups (p < 0.0001 and p = 0.0003 respec-tively). The median of the total lysozyme in the unstimulated saliva of NT and D as well as M and P groups were not significantly different from one another (Fig. 1B).
Lactoferrin
The median of the total lactoferrin in the un-stimulated saliva of NT group was significantly higher in comparison to D group (p < 0.0001) and it was on the same level in comparison to M and P group. The median value of lactoferrin in the un-stimulated saliva of D group was significantly low-er in comparison to M and P groups (p = 0.0004 and p < 0.0001 respectively). In the M group, the median value of lactoferrin in the unstimulated saliva was significantly lower in comparison to P group (p = 0.047) (Fig. 1C).
The Unstimulated Saliva
Flow Rate
The median value of the unstimulated flow rate in D group was significantly lower in
com-parison to M (p = 0.03) and P (p = 0.01) groups, whereas it did not differ significantly between these two last groups (Fig. 1D).
Stomatological Findings
There were no significant differences for dft index between D and M groups as well as for DMFT index between M and P groups. There were no significant differences for GI and API indexes between D, M, P groups (Table 1).
There were no correlations between dft, DMFT, GI, API, and salivary flow, the total amount of Lz and Lf and the specific activity of Px.
There was a correlation between the age and the total amount of Lz (r = 0.7, p < 0.0001) and Lf (r = 0.26, p = 0.04) and the specific activity of Px (r = 0.69, p < 0.0001) for all patients.
Discussion
The aim of the present study was to investigate age-related differences in salivary innate defence factors and salivary secretion.
In our study, an age-related increase was noted in the unstimulated salivary flow rate of the mixed and deciduous groups (p = 0.03). We assume that the salivary glands of the children from D group are not fully developed and have not reached their maximal secretory capacity, which is consistent with the results of Watanabe and Dawes [16]. This incomplete development of the salivary gland could be caused by immaturity of water secretion mech-anisms or smaller number of saliva secretion ele-ments (younger age of the child). It was shown that saliva is secreted from birth; flow rate increases with age due to a maturation of the salivary glands [17]. The lower salivary unstimulated secretion in chil-dren from D group suggests (despite the lack of correlation between DMFT and salivary flow) that children in this group are more likely to develop tooth decay and diseases of the oral mucosa than in other age groups. As is well known flushing effect of saliva is in fact the most important protective factor for oral mucosa and teeth against infectious diseases. Taking into account other risk factors of the development of dental caries (e.g. weaker min-eralization of deciduous teeth), this group should be subject to special preventive dentistry. No statis-tical differences in the salivary flow rates between the children with mixed and permanent dentition may suggest that mechanisms governing the water secretion in the salivary glands are fully developed already in the time of mixed dentition.
The contribution of the saliva to the preven-tion of oral diseases is due not only to the
con-stant flow of saliva, which mechanically removes the endogenous and exogenous microorganisms. A steady flow of saliva provides a continuous pres-ence in the oral cavity of proteins with protective properties. One group of such proteins is the pro-teins belonging to the innate immune system.
LF present in the saliva of breast-fed in-fants is derived largely from mother’s milk [18], which would explain the significantly higher to-tal amount LF in the saliva of toothless children compared to children with deciduous teeth, who were no breast-fed. We also observed a
gradu-al significant increase in totgradu-al amount of LF be-tween groups D and M and M and P. It is inter-esting that the total amount of LF in the saliva of toothless children did not differ significantly from group M and P. It can be said that the total amount of LF present in whole saliva seems to be at the adult level when no teeth are present. Per-haps this is a kind of adaptive mechanism protect-ing the oral tissues of the youngest children, when the adaptive immune system is still under devel-opment. The total amount of Lz did not differ be-tween groups NT and D, despite the fact that
ly-Fig. 1. The total content (amount of protein/100 mg of total proteins) of A) peroxidase (Px) (IU/100 mg of proteins),
B) lysozyme (Lz), C) lactoferrin (Lf) and D) salivary flow (mL/min). NT – tooth less group, D – children with decidu-ous dentition, M – children with mixed dentition, P – children with permanent dentition; p < 0.05 (Kruskall-Wallis test)
Ryc. 1. Całkowita zawartość (ilość białka/100 mg białka całkowitego) A) peroksydazy (Px, IU/100 mg białka
całkowitego), B) lizozymu (Lz), C) laktoferyny (LF) i D) przepływ śliny (ml/min). NT – grupa dzieci nieuzębionych, D – dzieci z uzębieniem mlecznym, M – dzieci z uzębieniem mieszanym, P – dzieci z uzębieniem stałym; p < 0,05 (test Kruskala-Wallisa)
sozyme is, similar to LF, provided to a child with his mother’s milk [18]. We did not note signifi-cant differences in the total amount of Lz between groups M and P. A significant increase in the to-tal amount of Lz occurred only between groups D and M. Both LF and Lz are synthesized by the cells of the salivary glands ducts [11] as well as they are released from the cellular components of the blood. An important source of salivary lactoferrin is secondary granules of neutrophils and its ex-tracellular concentration is considered as a mark-er of the activity of these cells [19]. Lysozyme, as about 25% of all proteins secreted by activated macrophages, can be considered as an indicator of the activity of these cells [20]. Despite the fact that in our study we did not observe significant correlations between the GI index and the total amount of LF and Lz, one must bear in mind: the process of tooth eruption. Tooth eruption leads to the development of a gingival crevice and to the increase permeability of the epithelium covering the erupting teeth, which may cause plasma de-rived LF and Lz to emerge and then mix with that secreted by the salivary glands [21]. The level of Lf and Lz can, therefore, be the result of the mat-uration of the salivary glands, as well as the pro-cesses accompanying the eruption of teeth. It was documented that the concentration of albumin in the saliva was found to significantly increase one month before the eruption of the infant’s first teeth, thus being a marker of forthcoming tooth eruption [21]. In our study, only lysozyme can be considered as an indicator of the first permanent teeth eruption, which may suggest that the ap-pearance of the first permanent teeth is accompa-nied by an increased inflow or increased secretory activity of macrophages.
Peroxidase is synthesized only in the salivary glands, mainly in the parotid gland. Its level in the saliva may thus be an indicator of the maturation of the structures responsible for its synthesis in the
salivary glands [1, 3]. In our research we have seen a significant increase in the specific activity of Px from NT to M group. The maturation process of the salivary glands, or rather their functions relat-ed to the secretion of proteins, seem to be complet-ed in the first stage of the emergence of permanent teeth in children with mixed dentition.
There is no data on the changes in the total amount of protein responsible for innate immu-nity of saliva depending on the age of child, so it is difficult to compare our results with the results of others. Tenovuo et al. [22] claimed that with the exception of lactoferrin and myeloperoxidase, the salivary innate defense system is at the adult lev-el when primary teeth erupt. Sonesson et al. [23] examined another innate proteins: MUC 5B (high molecular mucin) and MUC7 (low molecular mu-cin) in minor salivary glands secretions. They ob-served no significant differences between 3 years old, 14 years old and adults, however MUC7 in-creased significantly with age. They suggested that the mucin composition of the saliva varies in the developing child. It was also shown that in the whole saliva, the IgA concentration increases from childhood to adulthood [24, 25]. Other research-ers have observed significant decreases in the total mucin concentration among older people [26] and also a decrease in sialic acid concentrations dur-ing early life and agedur-ing [27]. All presented results demonstrate the variability of saliva immune sys-tems depending on age.
Recent studies have shown that the quality of innate immunity in the saliva of children de-pends on the age of child. Examined proteins are responsible for the homeostasis in the oral cavity, especially microbial balance. Perhaps changes ob-served in the present study in the total value of lac-toferrin and lysozyme and in the specific activity of peroxidase in the saliva may have implications for bacterial colonization in the children’s and ad-olescent’s oral cavity.
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Address for correspondence:
Anna Zalewska Skłodowskiej M.C. 24a 15-274 Bialystok Poland Tel./fax: +48 85 745 09 61 Mobile: +48 606 61 42 45 E-mail: annazalewska74@yahoo.com Received: 16.05.2013 Revised: 28.06.2013 Accepted: 18.07.2013
Praca wpłynęła do Redakcji: 16.05.2013 r. Po recenzji: 28.06.2013 r.
