PROMENE PLJUVAČKE KOD OSOBA SA BULOZNIM LIHEN PLANUSOM SALIVARY HUMORAL CHANGES IN ORAL BULLOUS LICHEN PLANUS

Cilj: Cilj istraživanja bio je ispitivanje humoralnih mehanizama u pljuvački kod pacijenata sa buloznim lihen planusom u fazi egzacerbacije i remisije. Materijal i metod: Praćeno je devetnaest pacijenata sa oralnim buloznim lihen planusom. Pljuvačka je prikupljana u jutarnjim časovima, bez stimulacije, u količini 5-10 cm. Određivanje imunoglobulina, C3 i C4 u pljuvački vršilo se primenom tehnike „microelis“. Cirkulišući imuni kompleksi u serumu i pljuvački određivani su PEG metodom. Dobijeni rezultati upoređivani su sa kontrolnom grupom i međusobno u fazi egzacerbacije i remisije. Rezultati su statistički obrađeni Studentovim „t“ testom. Rezultati: Ispitivanja su pokazala da je količina IgA u pljuvački značajno smanjena , dok je količina IgG i IgM značajno uvećana. Vrednosti imunoglobulina A , G i M u fazi remisije značajno su povećane kod bolesnika u odnosu na kontrolnu grupu. Upoređivanjem pljuvačke u fazi egzacerbacije i remisije dobile su iste količine IgG u oba stadijumima bolesti , ali evidentno povišene vrednosti IgA i malo značajno povećanje vrednosti IgM. U kontrolnoj grupi i grupi pacijenata u fazi egzacerbacije i remisije takođe su povećane CIC vrednosti . Podaci ukazuju na visoko značajnu depresiju komponente C3 u fazi egzacerbacije, dok je u remisiji vidljivo malo značajno smanjenje. Komplement komponente C4 u fazi remisije opada u poređenju sa kontrolnom grupom. Zaključak: Postoji evidentno učešće pojedinih komponenti pljuvačke u patogenezi oralnog lihen planusa, ali konačna pozicija za prevlast humoralnih mehanizama u patogenezi buloznog lihen planusa još uvek ne postoji.


Introduction
Oral lichen planus (OLP) is a chronic inflammatory disease, which mostly occurs in the middle -aged women 1,2 .Its etiology is unknown with possible multifactorial genesis 1 , such as stress, genetic predisposition, diabetes mellitus, or gastrointestinal disorders 3 .Clinical and histological studies highlight the role of some dental restorative materials such as amalgams 4 , composites 5 , and dentalacrylate 6 as the possible causes of this disease which very often has possible malignant transformation.The severity of the disease and the unknown nature require a constant study of etiopathogenetic activities during the course of the disease.
However, in recent years, it has become apparent that the immune system has a primary role in the emergence and evolution of oral lichen planus.In this regard, it is believed that salivary immunoglobulin levels may play a role in the pathogenesis of oral mucosal diseases, including lichen planus, or clinical changes which can affect the very disease 7 .In connection with this possibility, increased levels of serum immunoglobulins IgA and IgG were found in patients with OLP 8,9 .
Ghalayani 10 showed significant differences in the distribution of IgG + cells in different locations of oral lichen planus or lichenoid lesions separately, but the differences in the distribution of IgG + cells between oral lichen planus and lichenoid lesions did not prove as significant.Although Sistig 7 proved the increased levels of salivary IgA and IgG in these patients, der Waal 11 did not prove this assumption.
Considering the numerous inconsistent and controversial views, we set out to examine the salivary humoral mechanisms in patients with bullous lichen planus in the phases of exacerbation and remission, hoping to participate in clarifying the complex etiopathogenetic mechanisms of this very common disease.

Material and Methods
To realize this goal, at the Clinic for Oral Pathology and Periodontology, the Faculty of Dentistry in Skopje, 19 patients of different sex and age were followed, diagnozed with oral bullous lichen planus, regardless of topographic distribution of changes.
The study did not include patients with dermal manifestations, and with skin and oral presentation.Diagnosis was made based on a detailed history taking and objective clinical findings.A sample of saliva was taken from each patient.
Saliva was collected in the morning by a simple overflow without stimulation in the amount from 5 to 10 cm 3 .Then, no later than two hours of collecting, the material was transferred to the Institute of Transfusion where it was frozen and further processed.
All patients were assessed for immunoglobulin A, G and M, CIC, and complement components C 3 and C 4 .The tests were conducted twice in the phases of exacerbation and remission.The determination of immunoglobulins in saliva was performed using micro-ELISA technique.
The determination of C 3 and C 4 complement components in saliva was done in the same way as determination of immunoglobulins, except that the partigen plates have specific antibodies to these components.Normal values for C 3 range from 0.80 to 1.40 g / l and for C 4 from 0.2 to 0.5 g / l in serum.CIC determination in serum and mixed saliva was performed by the method of PEG (polyetilen glycol).The results were compared with a control group and with each other in phases of exacerbation and remission.
The control group comprised 25 healthy individuals, who did do not suffer from lichen planus or any other disease.The results for the values of immunoglobulin A, G, M, CIC and C 3 , C 4 in the saliva were compared with the control group.
All results obtained from comparative trials were compared with: the control group and each other in the phases of exacerbation and remission of the disease.
The results were statistically processed by applying the Student's t-test for the significance of differences in values.

Results
The results obtained in this study are shown in tables 1-5.
Salivary values for all analyzed immunoglobulins ( IgA, IgG and IgM ) in the examined and control group indicate statistical significance, whereas the value of IgA showed a significant decrease, and Ig G and Ig M significantly increased.
The values of salivary immunoglobulin A, G and M in patients with bullous lichen planus in the phase of remission are significantly increased compared with the control group ( p<0.001 ).
Comparative analysis of salivary immunoglobulins in the phase of exacerbation and remission points to identical amount of IgG in both stages of the disease, but an evidently increased value of IgA ( p<0.001) and statistically significant low elevated values of IgM ( p<0.05).
Salivary concentration values of the CIC in the control group of patients and patients in the phase of exacerbation and remission confirm the increase of all examined values ( p<0.001).
The data suggest a highly expressed significant depression of C 3 component (p<0.001) in the exacerbation phase, while in the stage of remission a low significant decrease is evident ( p<0.05 ).
Complement component C 4 in the bullous form in the phase of remission compared with a control group is declining (p<0.001).In the remission phase, complement component C 4 in saliva shows insignificant reduction ( p<0.2 ).

Discussion
Although previous in vitro studies indicated a serious participation of cellular immunity, they do not provide a definitive and convincing description of its activity in the course and pathogenesis of oral lichen planus 4,8 .
On the other hand, studies about the participation of humoral immunoglobulins, and serum complement resulted in inequality and contradiction 7,11 .Due to these inconsistencies, it is considered that there is no adequate information whether lichen planus is influenced by changes in the humoral or cellular immune response.Knowing the origin and properties of salivary immunoglobulins, it is natural to expect a change in their concentration in various diseases that attack oral cavity, including lichen planus.
IgG 4 in the exacerbation phase.At the stage of remission, IgG 1 and IgG 4 returned to normal, IgG 2 remained increased, which partly coincides with our findings.
Experimentally and theoretically is demonstrated that immunoglobulin A is a major protein whose main function is protective, directly related to the defense mechanism of the oral mucosa.Discounted values of this immunoglobulin are explained with the reduced resistance of the oral mucosa to numerous exogenous or endogenous factors, leading to certain pathological conditions, manifested up to possible classic, utterly benign, till the erosive-ulcerous lesions, with many dramatic clinical features, even with the possibility of malignant alteration.Examining the salivary biomarkers, Lopez-Jornet 12 suggests a possible function of oxidative stress that causes humoral salivary variations that affect the oral mucosa.
Parichehr Ghaleyani 13 relies on salivary immunoglobulin findings in clarifying the pathogenesis of bullous oral lichen planus, but argues that using a direct immunofluorescence can detect immunoglobulins and complement components in tissue samples from lesions of oral lichen planus, as useful parameters in the diagnosis of the disease.Sano 14 obtained similar findings, which emphasizes the fact that its use helps to clarify the physiology of bullous diseases which include the bullous OLP.
Generally, it is said that this immunoglobulin in the exacerbation phase is produced in larger quantities, through spillover of serum, or as a result of its local synthesis of the salivary glands.However, the capacity of this immunoglobulin to activate the complement, to bind to the antigen stimulator, and to make the association with CIC in the saliva, enabling reduced salivary values 7,13 and low salivary values of C 3 which are incorporated in this complex, is in fact our interpretation of the obtained results, typical of this stage of the disease.
The ability of this immunoglobulin is to increase the phagocytic activity of macrophages and indirectly to act on bacteriolysis.IgA realizes its function in the presence of complement and lysozymes, so it is logical and expected to find the low levels of complement component C 3 in our research.Dominantna komponenta u kliničkom ispoljavanju bulozne forme oralnog lihen planusa je prisutvo enantema i udružene eksudacije, u čijoj su osnovi vaskularna neravnoteža, prvenstveno naglašena filtracija kapilara i naglašena transudacija ali i proteini koji su poreklom iz seruma.Na osnovu ovih nalaza se može reći da je povećana koncentracija imunoglobulina A u pljuvački kod posmatranih grupa znak hemodinamskih varijacija.
The dominant component in the clinical manifestation of the bullous form of oral lichen planus is the presence of erythema and associated exudation, in which basis are the vascular imbalance, primarily emphasized capillary filtration and marked transudation, not neglecting even those proteins that come from serum.Based on these findings, we believe that the increased concentration of immunoglobulin A in saliva in the studied group are due to the underlying hemodynamic variations.
In the remission phase, one of the many features of IgA is to assist phagocytosis, to activate the complement through C 3 b component production, when the activation of the pathological development begins.The constant invasion of antigen material stimulates a continuous production of new immunoglobulin fractions, including IgG.One part of them, activated through the pathological processes, mediated by the complement system, makes a link with antigen, building CIC.
However, considering the increased biological effectiveness and suitability of alternative path and speed of a cascading process, starting with the C 4 component versus classical pathway, it is natural to expect a higher value compared with immunoglobulin A, whose values are reduced in the phase of exacerbation.If you add the growing need of the body for defense, then there is justification for our obtained results in terms of the value of IgG and received increased salivary values in all clinical forms in both stages of the disease [15][16][17] .
Overproduction of immunoglobulin M is directly associated with the acute phase of the disease.It is primarily involved in defending the body at the level of the mucous membrane in the early stage of the disease, the so-called stage of exacerbation.Persistent inflammatory reaction and secondary infection stimulate local synthesis of this immunoglobulin, which actually explains the the results obtained in this study.Immunoglobulin A is the main antibody in all secretions and in saliva, which is fully responsible for the implementation of the defense mechanisms of mucosal surfaces.Although the priority is here given to this immunoglobulin, other carriers of humoral immunity, immunoglobulin M and G should not be neglected 18,19  Prema ulozi medijatora u odgovoru na sistem C 4 komplementa, logičan je i očekivani pad vrednosti u fazi egzacerbacije i u fazi remisije bolesti.Ovaj proteolitički enzim je sposoban da nastavi odvijanje reakcije, delujući direktno na komponentu C 3 .
Given identical values of immunoglobulin G in the phase of remission and exacerbation in the bullous form, we assume that this immunoglobulin has not a crucial function in the saliva as immunoglobulin A. Evident increase in the immunoglobulin G in the stage of remission, versus the control group, and increased salivary immunoglobulin M, compared with control, probably is an additional factor in the effectiveness of salivary immunoglobulin system, aimed at defending the body at the level of the oral mucosa.We believe that these two immunoglobulin fractions have accessory role in the defense mechanisms of IgA immunogenic events in the oral mucosa.
CIC analysis in the phases of exacerbation and remission indicated an elevated value compared to the control group, whereas the values of the complement component C 3 are reduced in all patients in both phases of the disease.The interpretation of these findings is analogous to the former.As for the values of C 4 component in the saliva in phase of exacerbation, they are low.In addition, the difference between the bullous form and the control group is highly significant (p<0.001).In the remission phase, component C 4 in patients, compared to the control group, is easily reduced, so it is statistically insignificant.
According to the role of mediators in the response to the C 4 complement system, a decrease in the value is logical and expected in the phase of exacerbation and in the remission phase of the disease.This proteolytic enzyme is able to continue the progress of the reaction, acting directly on the component C 3 .
We assume that this component is installed in the CIC, and therefore its value declines in the circulation, analog to component C 3 .Chronicity of oral lichen planus and the fact that lichen planus in the largest percentage of patients is present till the end of their life is something that tells about its constant activity and function.

Conclusion
There is still no concensus on the dominance of the humoral mechanisms.It could be postulated that the involvement of certain components from the saliva in the pathogenesis of oral lihen planus is evident.

Table 1 .
Salivary values of immunoglobulin A, G and M in control and examined group in the phase of exacerbation

Table 2 .
Values of salivary immunoglobulin A, G and M in the control and examined group in the phase of remission

Table 3 .
Values of salivary immunoglobulin A, G and M in the examined group in the phase of exacerbation and remission

Table 4 .
Values of salivary immunoglobulin A, G and M in the control and examined group in the phase of exacerbation and remission Salivarne vrednosti koncentracija komplemenata C 3 i C 4 pljuvačke u kontrolnoj i ispitivanoj grupi u fazi egzacerbacije i remisije

Table 5 .
Values of salivary concentration of C 3 and C 4 complement components in the control and examined group in the phase of exacerbation and remission .