Gavrilova N.A., Gadzhieva N.S., Ivanova Z.G., Sapozhnikov A.M., Kanevskiy L.M., David А. Howe
Despite the wide range of medical and surgical methods of treatment used in ophthalmology, their effectiveness remains low in inflammatory processes in uvea – uveitis, often occurring with an autoimmune component. In the pathogenesis of uveitis plays the role of the immune reactivity of the organism, which includes links innate and adaptive immune responses.
The innate immune system forms a first line of defense towards pathogenic agents, penetrating into the human body [9, 13, 14]. The receptors of the innate immune system in comparison with the adaptive immune system are more conservative, their diversity is limited and is the result of phylogenesis. The answer of the innate immune system is developing rapidly and can lead to a rapid elimination of the pathogen without the participation of аdaptive immune responses.
Innate immune responses are realized through cellular and humoral factors. The factors include the nonspecific humoral immune defense – interferons, interleukins, chemokines, complement system, natural IgM and IgG antibodies and cellular factors – Toll-like receptors, cytokine receptors, natural killer cells – NK-cells, monocytes and macrophages, dendritic cells [16, 17] .
To identify the pathogenic invasion innate immune system of mammalian uses two methods – recognition of molecular structures of infectious origin – pathogen-associated molecular pattern (PAMP), the components of the cell of bacteria and fungi, lipopolysaccharides, lipopeptides, lipoproteins, peptidoglycan, β- glucagon or microbial nucleic acids or proteins (flagellin profilin); the second method – recognition of endogenous factors that arise in response to infection, recognition of "changing their" – distress associated molecular patterns (DAMP). Endogenous activators of innate immune responses include heat shock proteins, and uric acid, as well as products of necrosis and apoptosis [10, 11].
The pattern recognition receptors participate in the PAMP and DAMP recognition (PRR), in particular Toll-like receptors (TLR) that recognize various classes of microorganisms and provide an individualized response of the innate immune system to different types of infections. Furthermore, PRR exercise their influence on adaptive immune responses by dendritic cells whose main function is the induction and regulation of T-cell responses [11, 19].
TLR are pathogen recognition receptors and are considered by researchers as key receptors of innate immunity [6, 10, 11, 17]. TLR involved in the recognition of microbial components and initiate the activation of intracellular signaling pathways resulting in the expression of genes of cytokines (TNF, IL-1, IL-6, IL-12, and IFNα/β), costimulatory molecules and other genes.
A common feature of all toll-receptors is their ability to interact with the structures of bacteria, fungi, viruses, and to carry out an activation signal to the nucleus, leading to an increase in non-specific defense mechanisms of the body, in particular the inflammatory response, which leads ultimately to death and elimination of the pathogen. Toll-like receptors are expressed in immune competent tissues (tonsils, lymph nodes, spleen, thymus) and leather, broncho-pulmonary, gastrointestinal and urogenital epithelium, the epithelium of the cornea and conjunctiva, perivascular spaces of the iris and ciliary body. At the cellular level, Toll-like receptors are widely expressed on structural cells (epithelial, fibroblasts, endothelial) and immune cells – monocytes, macrophages, neutrophils, dendritic cells (DC), natural killers (NK-granular lymphocytes of the peripheral blood and lymphoid organs) and to a lesser extent on eosinophils, lymphocytes [6, 10, 12]. Between Toll-like receptors and interferon system there is a relationship – five types of TLR (3, 4, 7, 8, 9) are involved in the induction of biosynthesis of the three major classes of interferons [10, 11].
In a population of antigen-presenting cells of the iris, ciliary body, choroid human the study revealed high levels of expression of TLR-4, which are associated with the bacterial lipopolysaccharide receptor complex by the co-receptor – CD14 / MD-2-complex [1, 8, 9, 15]. In the normal human uvea the TLR-4 / CD14 / MD-2 complex is presented relatively abundant in the root of the iris and ciliary body stroma, whereas in the choroid and iris stroma it is detected rarely [3-5]. TLR-4 / CD14 / MD-2 complex in uveal tissue is localized perivascular and subepithelial. In contrast to other tissues such as skin, this complex is not expressed in the epithelium and vascular endothelium of the uveal tract of the eye [5, 13, 14]. Such localization of Toll-like receptors in the uveal tissue is the most optimal for binding to the gram-negative bacterial lipopolysaccharide complex in blood-ocular barriers . The Localization of the LPS receptor complex within the uvea involves a response reaction only by a penetration of microorganisms through the blood-and-tissue barriers.
The sensitivity of the PRR should be maintained at a level that would provide an enough reliable detection of microbes, but, if possible, minimize tissue damages. An uncontrolled activation of the PRR, including the Toll-like receptors, is potentially dangerous for the human, because it can lead to hyper-inflammation [9, 11]. Therefore, a negative regulation of pattern recognition receptors could, on the one hand, inhibit the PRR function and prevent the proper response of the innate immune system and, on the other hand, play a significant role in the treatment of pathologies resulting from hyperactivation of these receptors [5, 11].
To study and carry out comparative characteristics of expression of different types of Toll-like receptors on peripheral blood cells in the control and uveitis of various genesis.
Material and methods
The study involved 15 patients (7 male and 8 female) aged 25-54 years, who were treated for acute or exacerbation of chronic uveitis of various etiologies at the S. Fyodorov Eye Microsurgery Federal State Institution. The research carefully studied the history of the disease, the onset of disease, periods of exacerbation, results of treatment. Patients underwent blood ELISA to assess the nature of the pathogen, the process activity and immunological status. The status of the eyes was evaluated by standard examinations (biomicroscopy, direct and indirect ophthalmoscopy) and additional methods of investigation (the B-scanning of the eye, optical coherence tomography of retina and optic nerve). The control group consisted of healthy persons aged 23-27 years – 12 individuals.
Blood in an amount of 15 ml were taken on an empty stomach in heparinized sterile test tube. Peripheral blood mononuclear cells (PBMC) were isolated from blood samples by centrifugation on Ficoll density gradient. Toll-like receptor expression was measured by flow cytometry. Surface staining with FITC-labeled antibodies to TLR2, 4, 6 or intracellular staining of paraformaldehyde fixed cells with FITC-labeled antibodies to TLR3 and 9 was performed (all antibodies were from HyCult Biotech, The Netherlands). Samples were analyzed on FACScan cytometer (Becton Dickinson, USA), no less than 10000 events was recorded.
The statistical analysis was performed using the program "Biostat" by Mann-Whitney test for nonparametric data comparison of the two groups. The P <0.05 is a statistically significant result for all groups.
According to the localization of the inflammatory process, patients were distributed as follows: acute anterior uveitis or iridocyclitis were observed in 10 cases (11 eyes); panuveitis occurred in 5 cases (10 eyes), complicated by traction retinal detachment in three eyes, neuroretinitis – in 3 eyes, peripapillary exudative retinal detachment – in one eye. In 2 eyes of two patients the uveitis ended with the subathrophy of the eye. In 5 cases the inflammation was observed in both eyes.
Taking into consideration the polymorphism of clinical manifestations, etiological factors and the results of flow cytometry, we carried out a systematization of the results of the study. From the analysis of the study results 1 patient with keratoiridociclitis and 2 patients with progressive course of severe panuveitis complicated with subathrophy of the fellow eye were excluded; 1 patient with iridocyclitis autoimmune with diabetes mellitus, 1 patient – with hypopion-iridocyclitis in the pseudophakic eye, and 1 patient with recurrent iridocyclitis of an undetermined genesis. The results of the study of these patients were described as clinical case reports.
Comparative characteristics of the expression of various kinds of Toll receptors on peripheral blood cells in the control and uveitis of various etiologies are presented in the Tables 2-6.
The investigation results indicated that we watched a tendency to a significant decrease in expression of all types of TLR (2, 4, 6, 3, 9) on lymphocytes, monocytes and neutrophils as compared to the control. Statistically significant results were obtained by reduction of TLR2 expression on B lymphocytes and monocytes (p=0.02 and 0.014, respectively) more than 2 times; TLR6 and TLR4 – on monocytes (p=0.01 and 0.047 respectively). A trend toward greater reduction in expression of TLR 2, 4, 6, more than 2 times and less pronounced decrease TLR 3 and 9 (less than 2 times) for cells of the peripheral blood (Table 2-6).
Results of the study blood samples of 6 patients are presented separately.
The patient G., female, 26 years old, with severe type 1 diabetes, characterized an autoimmune multiple organ lesions, including bilateral iridocyclitis, there was an increase in the expression of all types of TLR (2, 4, 3, 6, 9) on lymphocytes; decrease in expression of TLR2 and 4 on monocytes and neutrophils; increase in expression of TLR6, 3, 9 on monocytes and neutrophils. Subsequently, the patient died of diabetic nephropathy.
Two patients with bilateral panuveitis progressive course, complicated by subathrophy one eye, saw an increase in the expression of TLR2, 6 on lymphocytes, monocytes and neutrophils, TLR3 – on lymphocytes, TLR4 – lymphocytes and neutrophils; decrease in expression of TLR9 – on all cell types, TLR3 – on monocytes and neutrophils.
Patient D., male, 50 years old, diagnosed with bilateral recurrent keratoiridocyclitis against the backdrop of chlamydial infection, marked reduction in the expression of TLR3 TLR2, 4, 6, 9 and increase on all types of peripheral blood cells.
The fourth patient was diagnosed with exacerbation iridocyclitis not installed genesis showed a significant increase, almost 2 times, all types of TLR on lymphocytes, monocytes and neutrophils.
Patient R, female, with a diagnosis of hypopion-iridocyclitis in the pseudophakic eye, we noted an increase in the expression of TLR on lymphocytes, TLR2, 3, 9 – on monocytes, TLR6, 3, 9 – on neutrophils; and decreased the expression of TLR4 on monocytes.
In summary, results of the present study demonstrated, that from all studied Toll-receptors we observed a significant inhibition of the expression of TLR2, TLR4 and TLR6 on peripheral blood monocytes. TLR2, TLR4 activated by lipopolysaccharide of Gram-negative and gram-positive bacteria through co-receptors (CD14 / MD-2 complex), their expression is increased under the influence of pro-inflammatory cytokines. TLR6 is activated by bacterial lipoproteins gram-positive and gram-negative bacteria, and functions as a heterodimer with TLR2 [5, 11, 15]. In the study group, where there has been a suppression of the expression of TLR2, TLR4 and TLR6, we watched uveitis of infectious-toxic recurrent nature (8 cases out of 9). Patients were treated with a steroid therapy. Inhibition of the expression of TLR2, TLR4 and TLR6 probably due to hyperstimulation of the toll-like receptor exogenous ligands before or at the onset of the disease and the development hyposensitivities of Toll-like receptors in the further development of the disease associated with the steroid therapy. In foreign literature, this state is called the «state of endotoxin tolerance» [7, 8, 19]. This condition is induced by glucocorticoids and in some cases is a defensive reaction from hyperinflammation and, according to the literature, it is transient. The sensitivity of the cells to stimulate PAMP restored within 48-72 hours [1, 11, 18].
In cases of progredient or overactive panuveitis (2 patients), we observed an increase in the expression of TLR2, 6 on lymphocytes, and neutrophils, TLR3 – lymphocytes, TLR4 – lymphocytes and neutrophils, which may indicate the hyperinflammatory response, involving the process all uvea. Iridocyclitis in autoimmune diabetes mellitus a progressive active inflammation with increased expression on B cells of all types of TLR (2, 4, 3, 6, 9) also took place; TLR6, 3, 9 expression was increased on monocytes and neutrophils. TLR2, 4 expression was reduced on monocytes and neutrophils. In our opinion suppression of expression TLR2 and 4 is connected in this case with the clinical state of the refractory as the protective reaction of the anterior segment of the uvea from hyperinflammation. The growth of the expression of Toll-like receptors on lymphocytes indicates the activity of the autoimmune process.
1. An infectious-toxic uveitis showed a significant inhibition of the expression of TLR2, TLR4, TLR6 on monocytes and TLR2 on B-lymphocytes in the peripheral blood. Inhibition of the expression of TLR2, TLR4 and TLR6 probably due to excessive stimulation of Toll-like receptors and development of state of the refractory Toll-like receptors in the active stage of the disease associated with an anti-inflammatory steroids therapy.
2. The greatest pathogenetic importance in the development of infectious-toxic uveitis has a dynamic change in the expression of TLR2, TLR4, TLR6 on peripheral blood monocytes.
3. Autoimmune uveitis showed a trend to increase the expression of TLR2, TLR4, TLR6, TLR3 on B-lymphocytes.
4. A further research of expression Toll-like receptors in an inactive stage of inflammatory processes of uvea is required as well as a comparative assessment of the nature of the expression of different types of Toll-like receptors in active and inactive stage of uveitis to evaluate the impact of inflammatory activity and drug therapy on the expression various types of TLR .
Сведения об авторах
Гаврилова Наталья Александровна, докт. мед. наук, профессор, зав. кафедрой глазных болезней;
Гаджиева Нурия Саниевна, канд. мед. наук, доцент кафедры глазных болезней;
Иванова Зоя Георгиевна, канд. мед. наук, доцент кафедры глазных болезней
ГБОУ ВПО «МГМСУ им. А.И. Евдокимова» Минздрава России
Сапожников Александр Михайлович, докт. биол. наук, профессор, зав. лабораторией клеточных взаимодействий;
Каневский Леонид Михайлович, канд. биол. наук, научн. сотрудник лаборатории клеточных взаимодействий
ФГБУН «Институт биоорганической химии им. акад. М.М. Шемякина и Ю.А. Овчинникова» РАН
David A. Howe, M.D.
Longevity Medicine LLC & Stemedica Cell Technolodies, USA