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Shadieva Shodiya Shukhratovna, researcher, of Tashkent institute of improvement of doctors
Alimov Alisher Sadikovich, doctor, of medical science, professor, of Tashkent institute of improvement of doctors E-mail: [email protected]
THE ROLE OF PROTECTIVE FACTORS OF BLOOD AND ORAL FLUID IN INFLAMMATORY PERIODONTAL DISEASES
Abstract: The aim of the study was to study some parameters of immune reactivity of the oral cavity in the development of lipopolysaccharide and immunocomplex inflammation in periodontal tissues. Thus, the differentiated approach of clinico-laboratory indices of lipopolysaccharide and immunocomplex inflammation of periodontal tissues showed significant changes in the studied parameters of the protective system of blood and oral fluid.
Keywords: lipopolysaccharide, immunocomplex inflammation, periodontitis, immunology.
Inflammatory periodontal diseases represent one of the topical problems in dental practice and are the main cause of premature tooth loss [4, 12]. Recently, numerous studies have shown that the inflammatory process in the tissues of the periodontium begins with the formation of dental plaques, mainly subgingival, as a result of colonization of the tooth surface by facultative anaerobes, primarily A. viscosus and S. Mutans [1, 8, 13]. In the initial colonization of the tooth, facultative anaerobes reduce the oxidation-reduction potential, thereby creating conditions for the reproduction of strict anaerobes, which are of great pathogenetic importance in the development of periodontal diseases [2, 9, 10]. Favorable conditions for the reproduction of anaerobic microorganisms are created in the depth of dental plaques, where up to 400 species of various microorganisms are isolated [12]. Many anaerobic bacteria are able to release toxic products and enzymes that have a cytotoxic effect, including collagenase, protease, hyaluroni-dase, neuraminidase, etc., capable of destroying the tissues of the macroorganism [6, 7]. In the further development of the process, enzymes secreted by phagocytic cells - lysosomal hydrolases and neutral proteinases of phagocytes, RNAase and DNAase - may participate. Macrophages accumulating in large numbers in the inflammatory focus are the producers of prostaglandin E2 and thromboxane B2, which are found in the tissues of the gums in periodontal diseases [5, 7]. The more severe course of the inflammatory process with periodontitis is explained by the fact that many microorganisms present in large amounts destroy IgA and IgG with their enzymes [3, 6]. By reducing the barrier function of the mucous membrane, these microorganisms facilitate the penetration and spread of toxic products, lytic enzymes and subgingival microflora in the periodontal tissues [11]. In the pathogenesis of periodontal diseases, an important role is played not only by microbial factors, but also by immunopathological mecha-
nisms - immunocomplex and cellular [12]. At later stages, an autoimmune component of the pathogenesis of periodontal diseases is added, which activates the complement system. Microbial antigens, cell decay products and dental plaque exchange products (chemoattractants) provoke an increased migration of segmented leukocytes and macrophages into the marginal epithelium. With the accumulation of specific antibodies (IgG and IgM), they form immune complexes with persistent antigens of a microbial nature, which should facilitate the purification ofthe mucosa from them [7, 14, 15]. The capture and degradation of immune complexes and their decay products are carried by migratory phagocytes activated by lymphokines into the inflammatory focus. If massive inflow of microbial antigens does not stop, mobilized protective mechanisms can lead to destruction of tissues. This is due to the release of phagocytic cells of lysosomal enzymes, among which neutral proteinases are most active: collagenase and elastase. They are capable of splitting the denatured collagen of the periodontal connective and bone tissues. In this case, the epithelium swells and loses a strong connection with the hard tissues of the tooth. One way or another, autoaggression mechanisms are formed that lead to a progressive, recurrent, irreversible course of periodontitis with atrophy of osteocytes and alveolar processes of the jaw [11].
Based on the above, the purpose of this study was to study some parameters of immune reactivity of the oral cavity in the development of lipopolysaccharide and immunocomplex inflammation in periodontal tissues.
Research material and methods. Subjects of the study were patients with chronic generalized periodontitis of moderate severity. 68 patients aged 37 to 55 years and 13 volunteers aged 23 to 32 years were examined. They turned for help to the clinic of the Department of Therapeutic Dentistry of Bukhara State Medical Institute. Clinical and laboratory stud-
ies were carried out using the equipment and apparatus of Bukhara State Medical Institute laboratories, as well as the immunological laboratory of the Institute of Immunology of the Russian Academy of Sciences.
Criteria for the inclusion of patients in the study: voluntary (written) and conscious participation in the study, the absence of severe concomitant pathology, the diagnosis of "periodontitis of moderate severity", orthognathic bite, the presence of permanent orthopedic structures, the absence of no more than two teeth on each side of the jaw, the presence of single crowns, women lack of pregnancy, menopausal syndrome, the absence of bad habits: smoking, alcoholism, obesity.
The criteria for exclusion from the study: periodontitis of mild and severe severity, oral diseases, pathological types of occlusion, systemic diseases, difficulties with reading and filling out questionnaires, occupational hazards, disagreement with the conditions of the study. Criteria and non-inclusion in the study: alcohol dependence, drug dependence, the presence of acute somatic inflammatory diseases, age less than 28 and older than 50 years.
Division of patients with the inflammatory process in periodontal tissues into groups was performed on the basis of clinico-laboratory signs of lipopolysaccharide and immuno-complex inflammation. According to clinical and laboratory indicators, three groups were identified: 1. Group - patients without pathology in the oral cavity (healthy); 2 Group - patients who have an inflammatory process in the tissues of the periodontal lipopolysaccharide etiology; 3. Group - patients with instrumentally confirmed inflammation of the immu-nocomplex etiology. The presence of a lipopolysaccharide inflammatory process in the examined subjects was assessed by the content of the morphological forms of the cellular elements of the leukocyte series in the peripheral blood (leukocyte formula), inflammatory mediators (interferon y-IFNy, pro- and anti-inflammatory cytokines). The presence of im-munocomplex inflammatory process in the examined subjects was evaluated by such indicators as components of the humoral link of the innate immune response (C-reactive protein,
complement components) and endothelial cell dysfunction (endothelin and von Willebrand factor).
Estimation of the periodontal tissue condition was carried out with the help of the simplified hygienic index (UGI) -ON1-B, the index of hemorrhage of the gingival sulcus IR-SBI, the periodontal index (PI) -P.1.R, the depth of the periodontal pocket was measured with a graduated punched periodontal probe. All patients underwent X-ray examination to determine the condition of the periodontal bone tissue.
The concentration of pro-inflammatory (TNF-a, IL-1a, IL-6, IL-8), anti-inflammatory (IL-4, IL-10) cytokines and complement components (C3 and properdin factor B) was determined in serum and mixed oral fluid with sets of reagents of "Vector-Best" Closed Joint-Stock Company and NGO Tsi-tokin in accordance with the methodology proposed by the manufacturer. Immunological studies were conducted jointly with the staff of the Research Institute of Immunology of the Academy of Sciences ofUzbekistan. A study was conducted: secretory IgA (IgA secretory-IFA-BEST, producer ofVector-Best CJSC, Russia); lysozyme (LIZOTSIM-IFA-BEST, producer ofVector-Best CJSC, Russia). Statistical processing of data was carried out using the Microsoft Excel 2016 package (Microsoft SOG, USA) and Statistica version 6.0 (StatSoft Inc., USA). The characteristic values included estimates of the mean, variance, coefficient of variation, standard deviation. Verification of hypotheses about the existence of significant differences between the averages was carried out using Student's t-test.
The results of the research and their discussion. Under the influence of the products of vital activity of parodonto-pathogenic microorganisms (PPM), the dentogingival joint is damaged, which leads to an increase in the permeability of the epithelium of the oral mucosa. A direct response is an intensive migration into the lumen of the dentogingival furrow of polymorphonuclear leukocytes and cells of the monocyte-macrophage system. The proliferation of polymorphonuclear leukocytes into the inflammatory focus occurs in several stages, the final of which is diapedesis of leukocytes between endothelial cells (transendothelial migration).
Table 1.- Absolute and relative number of subpopulations of immunocompetent cells in the examined subjects against the background of inflammatory periodontal diseases
Index Persons without paro-dontium pathology Lipopolysaccharide faces with inflammation of periodontal tissues Persons immunocomplex with inflammation of periodontal tissues
1 2 3 4
Leukocytes, -109/l 5.4 ± 0.8 8.1 ± 1.3 6.1 ± 1.2
Lymphocytes,% 35.5 ± 1.0 29.7 ± 0.9* 39.9 ± 1.1*
CD3+,% 62.4 ± 2.0 55.2 ± 1.6* 67.3 ± 1.5*
CD3+,-109 1.12 ± 0.1 1.05 ± 0.2 1.88 ± 0.1*
CD4+,% 43.9 ± 1.5 39.4 ± 1.4* 37.5 ± 1.6*
CD4+,-109 0.78 ± 0.09 0.75 ± 0.05 0.65 ± 0.04
1 2 3 4
CD8+, % 24.2 ± 1.3 26.1 ± 1.1 29.9 ± 1.2*
CD8+,-109 0.43 ± 0.04 0.49 ± 0.07 0.57 ± 0.06*
IRI, y.e. 1.81 ± 0.3 1.50 ± 0.2 1.69 ± 0.1*
CD16+, % 15.6 ± 0.9 17.5 ± 0.8 24.8 ± 0.4*
CD16+,-109 0.30 ± 0.01 0.32 ± 0.02 0.51 ± 0.03*
As can be seen from the presented research results, in patients with liposaccharide inflammation of periodontal tissues there was an increase in the number of leukocytes, whereas the level of the pool of lymphocytes was reduced when compared with a group of individuals without periodontium pathology (Table 1). The latter is accompanied by a decrease in the percentage of CD3+, CD4+. In the remaining indices of the absolute and relative number of subpopulations of immunocompetent cells in the examined subjects against the background of inflammatory periodontal diseases lipopoly-saccharide etiology was not observed. Interesting dynamics was noted with inflammation of periodontal tissues of immune complex aetiology, where an increase in the percentage of CD3+, CD8 and CD16+ was noted, while the percentage of CD4 + was decreased when compared with the control group. Consequently, the indices of cellular immunity in patients with inflammatory periodontal diseases are of a peculiar nature. In patients with inflammatory periodontal disease, there was a significant decrease in the immunoreactive index of CD4 / CD8 (1.50 ± 0.2) compared with healthy individuals (1.81 ± 0.3). Therefore, the inflammatory disease of periodontal tissues of lipopolysaccharide etiology is associated with disorders in the cell link of immunity: relative lymphopenia, imbalance of T-cell subpopulations with a decrease in
the relative values of T-helper content and an increase in the relative number of cells carrying the marker of natural killers (CD16). In individuals, inflammatory diseases of periodontal tissues of immune complex aetiology are associated with impairments in immune status; an increase in the percentage of lymphocytes, as well as populations of lymphocytes (CD3, CD8, CD16).
Against the backdrop of inflammatory diseases of periodontal tissues, the examined subjects noted disorders in immune mechanisms of oral cavity protection, manifested by local changes in the cytokine profile. One of the key links in the generalization of the pathological process in the periodontium is the mediator of inflammation of interleukin-1 (IL-1). IL-1 stimulates the production of adhesion molecules by endothelial cells, which facilitates the attachment of polymorphonuclear granulocytes and monocytes, as well as the mobilization of these cells into the inflammatory focus. Lipopolysaccha-rides of the cell wall of odontopathogenic bacteria stimulate macrophages to produce pro-inflammatory cytokines, further their production is amplified by an autocrine mechanism. In addition, leukotoxin A. actinomycetemcomitans can trigger the abundant secretion of bioactive IL-1p by macrophages. With the constant massive intake of microbial agents, the production of pro-inflammatory cytokines becomes excessive.
Table 2. - Cytokine profile of blood plasma and oral fluid in subjects undergoing a background of inflammatory diseases of periodontal tissues
Index Persons without paro-dontium pathology Lipopolysaccharide faces with inflammation of periodontal tissues Persons immunocomplex with inflammation of periodontal tissues
Blood
IL-1|, pg/ml 1.72 ± 0.05 1.98 ± 0.08 9.9 ± 1.2*
IL -4, pg/ml 0.39 ± 0.2 1.25 ± 0.3 2.03 ± 0.8*
IL -6, pg/ml 1.69 ± 0.09 2.59 ± 0.76 17.3 ± 2.3*
Tumour necrosis factor -a, pg/ml 0.57 ± 0.01 1.17 ± 0.2 36.5 ± 3.1*
IF-y, pg/ml 2.1 ± 0.4 2.8 ± 0.5 4.5 ± 0.7*
Oral fluid
IL-1|, pg/ml 3.6 ± 0.23 21.9 ± 1.5* 44.5 ± 4.2*
IL-4, pg/ml 26.5 ± 1.7 32.6 ± 1.9* 54.4 ± 2.7*
IL-6, pg/ml 3.05 ± 0.08 10.4 ± 1.1* 25 ± 1.6*
Tumour necrosis factor -a, pg/ml 15.4 ± 1.1 36.4 ± 3.8* 112 ± 4.2*
IF-y, pg/ml 39.4 ± 2.3 97.5 ± 5.3* 231 ± 4.9*
Proinflammatory cytokines also stimulate the production of matrix metalloproteinases, reduce the production of the tissue inhibitor of metalloproteinases, induce the activity of the cytokine bone remodeling system RANKL (Receptor activator of nuclear factor kappa-Bligand), and thus potentiate the resorption of the alveolar bone, and directly activate the osteoclasts. One of the powerful anti-inflammatory cytokines is IF-y, which blocks the autostimulation of pro-inflammatory cytokines by macrophages. However, an increase in its level, as well as the level of IL-4, was insufficient to limit pro-inflammatory cytokinemia. Those. with inflammatory periodontal diseases, the anti-inflammatory response is unbalanced, which leads to an inadequate immune response to the periodontal pathogens and to the prolonged course of the inflammatory process in the periodontal tissues.
Based on the foregoing, it can be concluded that the inflammatory process in the periodontal tissues is accompanied by a significant increase in proinflammatory mediators and a smaller increase in the content of anti-inflammatory cytokines both in the oral fluid and in the blood, i.e. had a systemic character. Revealed factual material can serve as an additional diagnostic and prognostic marker of the severity of inflammatory periodontal disease. Thus, the surveyed individuals with inflammatory periodontal disease noted a multidirectional change in the content of pro- and anti-inflammatory cytokines both in the blood and in the oral fluid. In this case, the change in cellular immune reactions is unidirectional and consists of a decrease in the relative
As can be seen from the presented research results (Table 2), all patients had significant differences in the degree of activity of the classical complement pathway, in particular, the content of the C3 complex component. The observed high activity of the classic complement pathway in the examined patients of groups with immunocomplex etiology may be associated with the need to resist tissue damage. Based on the aggregate of the data obtained in the study, it is suggested that the recorded continuation of the activation of the classical complement pathway in patients is associated with an increase in the serum of the titer of antibodies of class M and G, both endog-
number of the general population of lymphocytes, mature T-lymphocytes, T-helpers, along with an increase in the relative number of subpopulations of natural killers.
In the pathogenesis of inflammatory periodontal diseases, an important role is played not only by microbial factors, but also by immunopathological mechanisms - immunocomplex. At later stages, an autoimmune component of the pathogenesis of periodontal disease is attached. This gives rise to a specific immune response, immune inflammation. With an increase in the amount of Gram-negative bacteria and their destruction, endotoxin is released, the action of which increases the formation of IgM and reduces IgG. Antibodies related to IgM reacting with antigen-antibody reactivate the complement system. The complement system is one of the main effector mechanisms for the realization of both congenital and acquired immunity. And, despite the fact that all activation and regulatory mechanisms have not yet been determined, studies in this area demonstrate the importance of the complement system for various diseases. The results of the studies presented by us testify to the undoubted contribution of the components of the complement system to the pathogenesis of immunocomplex inflammation of the periodontal tissues. The resulting immune complex with IgG and IgM class antibodies is one of the reasons for the conformational changes and provides the start of a cascade of complement-dependent cytolysis, which is often complicated by damage to the peri-odontal tissues.
enous and microbial in nature. Microbial invasion could be realized due to the translocation of microflora from the focus of inflammation caused by endothelial dysfunction. Due to the adequate response of the innate immune system, bacterial invasion was not fatal, and presentation of the antigen to cells of the adaptive immune response allowed switching the immune defense to the next level, with the synthesis of specific antibodies, in particular the transition to the formation of immune complexes and indirectly, the connection to the process of inflammation of components adaptive immune response. However, it is possible that relatively high levels of activity of
Table 3.- Dynamics of the quantitative content of the complement component of the classical pathway of immunoglobulin M and G in the subjects (mg/dL)
Index Persons without parodontium pathol- °gy Lipopolysaccharide faces with inflammation of periodontal tissues Persons immunocomplex with inflammation of periodontal tissues
The complement component of C3 65.4 ± 4.81 78.1 ± 6.32 176.1 ± 10.24*
The complement component of C5a (pg/ml) Immunoglobulin M 2.33 + 0.11 155.5 ± 11.02 3.28 + 0.13* 99.7 ± 4.96* 5.86 + 0.44* 239.9 ± 17.12*
Immunoglobulin G 1062.4 ± 23.07 1255.2 ± 31.62* 1667.3 ± 43.54*
the classical pathway observed in the patients being examined indicate a sufficient severity of the immune response in this category of patients, which, apparently, is associated with the depletion of immune response reserves due to the progression of the underlying disease in the previous period.
Complement C5a is a multicomponent enzyme system of plasma, which manifests upon activation of the function of lysis and opsonization. So initially the amount of this component in healthy individuals was at the level of (2.33 + 0.11 pg/ml). In patients with the immunocomplex etiology of inflammation, its higher concentrations (5.84 + 0.44 pg/ml) were determined, which exceeded them by a factor of 2. It is known that the complement system C5a is always in a state of readiness, but its activity is minimal. This watchdog function is ensured by fixing the C3 subunit of complement on pathogens and the host's own tissues. Through this component of complement all three known ways of activation are realized: classi-
cal, lectin and alternative. When C3 is initialized through any of the pathways, the whole complement activation pathway progresses, resulting in the formation of anaphylactic C5a and terminal membrane-attack complex C5B-C9. Accordingly, high concentrations of C5a, determined in the group by the immunocomplex etiology of inflammation of the periodontal tissue, are predisposing to the formation of autoagression to the periodontal tissues. A prolonged increase in the levels of this subcomponent of complement potentiates the damaging effect of membrane-binding complexes and leads to traumati-zation of tissues, including the vascular endothelium.
Thus, the differentiated approach of clinico-laboratory indices of lipopolysaccharide and immunocomplex inflammation of periodontal tissues showed significant changes in the studied parameters of the protective system of blood and oral fluid, which is closely related to the degree of damage to periodontal tissue, the duration of the disease and inadequate therapy.
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