Журнал «Здоровье ребенка» 7 (50) 2013

Abstract

The paper presents an overview of research on the study of the question of apoptosis of immune cells in pathological processes, including at the Epstein-Barr virus infection. Also presented its own investigations which revealed a violation of the expression of markers of apoptosis of immune cells in children with acute EBV infection. Comparative study of apoptosis markers in children with acute EBV infection showed a high degree of expression. All the studied markers of apoptosis: Fas/Apo-1, Bax, INFγ, TNFα, annexin V, Bcl-2 exceeded the control values ​​by 2-3 times or more.

Introduction. The role of apoptosis in viral infections is of fundamental interest for the understanding of the pathogenesis of the immune system, has a practical value for the objective study on a patient. In recent years, this process is of interest c positions of molecular biology, biochemistry and genetics.Apoptosis is a form of cell death, which is detected by morphological identification: chromatin condensation, reducing the size of the cells, the fragmentation of the nucleus and cytoplasm, the formation of apoptotic cells [1, 2]. Most viruses are transmitted by airborne droplets, including Epstein-Barr virus ( EBV ) can induce apoptosis in the cells of the respiratory epithelium, and thus the process of apoptosis can be activated by tumor necrosis factor α (TNFα) - apoptosis-inducing ligand TNF-related apoptosis-inducing ligand (TRAIL), which leads to the selective destruction of virus infected cells [3-8]. TNFa is a soluble cytokine synthesized by activated T-lymphocytes and macrophages in response to infection and inflammation [9]. Its binding to TNF- receptors (TNF-R) leads to the same reaction as in Research binding Fas receptor (Fas-R), and Fas ligand (Fas-L or CD95L), with the only difference that - mobilized forms a protein TRADD (TNF receptor-associated death domain) [10, 11]. This in turn leads to increased transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) and the plasminogen activator-1 (AP-1) [12]. TNFα stimulates the adhesion of neutrophils to endothelial cells and their extravasation - migration to the site of vascular inflammation (facilitating loosening of the extracellular matrix), promotes the activation of neutrophils, enhancing phagocytosis and the production of superoxide radicals, as well as the expression of complement receptors on neutrophils, induces the expression of additional receptors Fas- on T-lymphocytes, human leukocyte antigen (HLA-DR) expression and IL-2 high affinity receptor (IL-2) [11, 13]. T- cells under the action of TNFα accelerate proliferation in response to IL-2 and increasing IL-2- dependent synthesis of interferon- γ (IFNγ). TNFα is an autocrine and paracrine activator of macrophages, enhancing their ability to kill bacteria. TNFα is a chemoattractant for macrophages and Langerhans cells in the skin, stimulates the production of IL-1 , Prostaglandin E2 (PGE2), granulocyte -macrophage colony stimulating factor (GM -CSF). TNFα acts mainly on the early stages of the inflammatory process. It is necessary for the completion of transforming growth factor beta (TGFβ) . The synthesis of TNFα has been declining for several hours after activation. TNFα has pro-inflammatory and katobolicheskoe strong action has antimicrobial and antitumor activity and interacts with the membrane receptor Fas cemeystvo (p55) [3, 9-12]. In some situations it is important for hard apoptosis regulation in maintaining the integrity of tissues and cell turnover functions, therefore, is generally considered as an anti-inflammatory process. The role of apoptosis in viral infections is limiting the scale of infection, including inflammation and is a common cellular response. Macrophage cells which phagocytose apoptotic cells become at this inflammatory properties. In such increased expression of TGFβ macrophages and PGE2, decreases production of IL-8, TNFα, IL-1β, monocyte chemoattractant protein-1 (MCP-1). Macrophages are phagocytosed apoptotic cells are capable of inhibiting the proliferation of T-lymphocytes, unlike macrophages phagocytosed necrotic cells [14]. Recently, much attention is paid to the study of the biological activity of proteins belonging to the family of annexin. Annexin V (Ann V) refers to a family of Ca +2 and fosfolipidsvyazyvayuschih proteins blocking phospholipase A. Ann V mechanism of action is very important property is its contact with the negatively charged phospholipids including phosphatidylserine (PS), whose exposure to the cell membrane is one of the earliest signs of apoptosis [ 4]. Annexin V, as well as other annexins not distinguished from normal cells, extracellular source Ann V are destroyed and apoptotic cells [3, 15]. Studies have shown that B-cell lymphoma 2 (Bcl-2) in - directly or indirectly prevents release of cytochrome C deniyu translocation gene Bcl- 2, leading to its overexpression, characteristic B-cell lymphoma, and certain forms of cancer resistance to chemotherapy [16 -20] . Antiinflammatory and stored function of protein Bcl -2 is to protect endothelial cells by inhibiting nuclear factor NF-kv and reduce production of proinflammatory genes [14, 21]. Proapoptotic protein Bax (apoptosis regulator BAX or bcl- 2 - like protein 4) moving from the cytosol to the mitochondrial surface, inactivate anti-apoptotic proteins leading to the formation of pores in the mitochondria, and release of cytochrome C and other proapoptotic molecules of intermolecular space [22]. Proapoptotic Bcl- 2 members also increase the permeability of the mitochondrial membrane, which leads to the penetration of proapoptotic protein in the cytoplasm. Like other members of the family, Bax has constant domains of Bcl-2 homolog 1 (HV 1) and 2 (HV 2), which determines their homo-or heterodimerization in Bcl-2. Although Bax that by itself does not induce apoptosis, increase the level of apoptosis Bax accelerates after death signals, such as cytokine deprivation or interaction with Fas- Fas ligand [23]. Is defined by the occurrence of apoptosis Bax homodimers and heterodimers Bax/Bcl-2.When the Bcl-2 in excess of apoptosis is inhibited. However, if increasing the level of Bax in response to the death, it promotes cell death. The sensitivity of the different classes of lymphocytes to apoptosis significantly different. Since T- cells are more sensitive to apoptosis than B lymphocytes [24]. This is because the antigen recognition receptor activation of T-lymphocytes leads to a drastic increase in sensitivity to apoptosis of cells, while at the same time antigen- receptor activation in cells causes resistance to apoptosis of lymphocytes. Despite the fact that activated B-cells are able to survive after the interaction with a specific autoantigen, they are not able to develop an immune response without the support of the T-helper cells.Data on the formation of annexin V, Fas/Apo1, Bax, Bcl-2, and TNFα,  intracellular INFγ with EBV infection are scarce and contradictory [ 25].

In view of the foregoing, the objective of our investigation was to study the state ofexpression in immunocompetent cells apoptosis markers Fas/Apo-1, Bcl-2, Bax, INFγ, TNFα, annexin V of undergoing apoptosis in the circulating blood of patients with acute EBV infection to clarify their role in disease pathogenesis.

Materials and methods

The study was conducted in 25 children aged 3 to 18 years, patients with acute EBV infection who were treated at the city children's clinical hspital of infectious diseases in Kiev from 2008 to 2012 years. The diagnosis of acute EBV infection established by history, clinical and laboratory tests (complete blood count, determination of transaminase activity of blood), the identification of specific markers of EBV infection - antibodies of the IgM VCA, IgG VCA, IgG EA to EBV , in the absence of blood IgG EBNA, determination of EBV DNA in the blood and saliva. Among children, boys were 45,0 % , 55,0 % girls , children from 3 to 6 years of age – 35,0 %, 7-9 years – 25,0 %, 10-14 years – 20,0 %, over 14 years – 20,0 %, with a moderate form of the disease was 50,0 % of the children of light – 25,0 % , severe – 25,0 %.

Analysis of complaints and objective examination of the data showed that all the children the disease begins acutely with intoxication syndrome, which manifested itself in the form of general weakness, malaise (100,0 %), decreased appetite (75,0 %), headache (83,3 %), nausea (50,0 %) in children with severe forms - arthralgia, myalgia, vomiting. The central nervous system (CNS ) is also observed changes in all the children in the form of emotional lability, tearfulness, irritability, negative reaction to the survey, sleep disturbance was detected in 66,7 % of patients. Rash occurs in 40 % of patients - mostly in children treated with ampicillin at home, or derivatives thereof. In most patients rash appeared on 4-7 days of treatment and was maintained for 10-14 days. Prevailed maculla-papular rash (61,9 %), medium and/or large, located all over the body, most patients rash wore drain character. In 23,8 % of the children had a rash rosella, in 14,3 % of patients - hemorrhagic. Hyperthermic syndrome was detected in all children. Increased body temperature to 38°C was observed in 37,0 % 38,1 - 39°C in 35,2 %, 39 - 41°C in 27,8 % patsientov. Lymphadenopathy found in all patients. The systemic nature of lymphadenopathy was at 76,7 % of children. Mainly been an increase in the submandibular (73,3 %), neck groups (83,3 %), inguinal groups (50,0 %) lymph nodes. Were also enlarged lymph nodes and other groups - axillary (50,0 %), occipital ( 45%), subclavian and supraclavicular (41,7 %). All patients had a lesion in the nasopharynx - nasal congestion – 86,8 % of children, facial edema and age – 52,8 %, difficulty in nasal breathing – 75,5 % , "snoring" during sleep – 50,0 %. Nasal discharge appeared after the 4th day of illness onset and occurred in 47,2 % of patients. All the children had a place of acute tonsillitis, which was manifested discomfort, pain in the throat when swallowing, hyperemia of the mucosa of the oropharynx, grain size of the soft palate, temples, back of the throat. At the time of hospitalization layering on the tonsils were observed in 81,7 % of patients. Of these, 65,3 % were purulent, at 34,7 % - filmy , and 25 % - cheesy. In 5 patients, accretions on the tonsils were not, they noted only redness and swelling of the mucous membrane of the oropharynx. Constant symptom of the disease was hepatomegaly, revealed in all children. The phenomena of cytolytic hepatitis syndrome was observed in one in three patients. In these patients, in the absence of hyperbilirubinemia recorded increased activity of alanine aminotransferase (70,5 ± 7,8 units), aspartate aminotransferase (59 ± 2,8 units) . This yellow skin and mucous membranes occurred only in 24,5 % of patients. These children have experienced moderate hyperbilirubinemia (90,0 ± 4,6 mkol/l). Splenomegaly was present in 86,7 % of patients. Hematologic abnormalities were observed in all children. In the blood of 73,3% of patients had mild leukocytosis - (14,4 ± 3,5 ˟10⁹/l), lymphocytosis – 88,3 %, monocytosis – 83,3%. In 61,7 % of the patients were found abnormal mononuclear cells, their number in peripheral blood ranged from 10 to 55 %, the ESR was accelerated in 70,0 % (25 ± 5,2 mm/h).

Reference values (n=25) were analyzed in the control group, which included 25practically healthy children at the age of 7 to 14 years who were screened in accordance with international ethical protocol.

Procedure of the study. The object of the study was immunocompetent cells (ICC) that were isolated from venous blood, stabilised with EDTA (50 mmol/5ml), in ficoll-verograffin gradient (1,077 “Pharmacia”). Between each stage of study, ICC were three-fold washed with PBS solution (pН 7.2; “Flow Labs”, UK). Then in neutral formalin vapour they were fixed on glass slides (50μl/cavity) at concentration 1-2 million/ml and stained using monoclonal antibodies marked with FITC or PE, PERCP, Су5; ICC nuclei were stained with Hoechst or Propidium iodide (PI). Results were calculated on luminescence microscope using the computer program Multichenel AxioVision 4.6. There was used confocal laser scanning microscope Axioskop-2 LSM 510 PASCAL Carl ZEISS with helium-neon and argon lasers (Lazer 405/488 /543/633 nm, filters BP 505-530, LP 650, BP 420-480). Objective – 100/1.4 160/017, ocular 10(23), oil immersion. Obtained images were scanned and processed using the computer program LSV510. Differential markers were determined using acridine orange conjugated to fluorescent probes with different emission spectra at Еm-max (FITC/519- green, PE/578- yellow, Cy5/667- red, PerCP/678- red).

Statistical treatment of the results were performed using methods of modern medical statistics. Fordata statistical processing, there was used MS Excel 2007 [26].

Results and discussion

Comparative study of apoptosis markers in acute EBV infection showed a high degree of expression. All studied markers of apoptosis: Fas/Apo-1, Bax, INFγ, TNFα, annexin V, Bcl-2 higher than the reference values​​, and 2-3 more times (p <0,05). In children with acute EBV infection on admission to the hospital level Fas/Apo-1 exceeded the reference value of 2,9 times, the level of Bcl-2 was increased by 2,3 times, the level of Bax - by 3,2 times, the level of INFγ - in 3,1 times, the level of TNFα - 3 times, the level of annexin V (Ann V) – 2,5-fold (p<0,05).

Role of apoptosis markers studied for EBV infection in children with one hand indicates compensating possible immune system by increasing the expression of Bcl-2 gene expression and gene amplification in wah subpopulations CD4 and CD8. Activation of the expression of proapoptotic TNFα receptor in the main pool of cells inducing the expression of Fas- additional receptors. High expression of Fas-R on the ICC in patients with MI EBV etiology of children c simultaneous activation of CD8 expression of differentiation markers (potentially carrying the Fas-L) indicate the direction of apoptosis to nitric oxide and Fas- mediating the way. Major role in the elimination of infected cells in patients with EBV infection of children perform a cytotoxic T -suppressors. Activation of expression of markers of apoptosis promotes the release of large amounts of nitric oxide, which induces the release of toxic substances for pathogens, contributing to increased intracellular apoptotic cells persist pathogens. Low amounts apopotoznyh cell population of T-helper dependent high expression therein antiapoptotic gene Bcl2, which keeps the cells from apoptosis. Another argument in favor of compensatory reactions of the immune system in myocardial infarction in children include the normal level of expression of intracellular INFγ, which is a reserve and macrophage cytotoxic activity in the anti-proliferative and anti-virus protection.In the available literature there are few works on the study of apoptosis in EBV infection. So, Zheleznikova GF et al. conducted a study of apoptosis in infectious mononucleosis EBV etiology in children and received data showing that increasing the severity of clinical symptoms of acute infectious mononucleosis in children is associated with a change in the profile of the leading mechanisms of immune protection, reduced expression of Fas/CD95 and a tendency to spontaneous apoptosis in vitro. The high response of T lymphocytes PHA RBTL, increased TNFa, relatively low level synthesis of IgA and IgE, accumulation CEC children with relatively mild infectious mononucleosis indirectly indicate predominantly cell-mediated immune response direction. In contrast, suppression of PHA RBTL , prevalence IL- 4 at a reduced concentration TNF-a levels, elevated blood CD21 + B lymphocytes and plasma cells, activated synthesis of IgA and, especially, IgE, increased accumulation of CIC in children with clinically more severe infectious mononucleosis suggest they shift the balance of Th1/Th2 toward Th2 predominance and humoral immune response forms. This is accompanied by a decrease in apoptosis receptor CD95 expression on circulating lymphocytes, and an increase in survival of cells in culture [25].The pathogenic significance of modulation immunogenesis and apoptosis in acute Epstein- Barr virus infection remains unclear, necessitating further research. It is possible that the decrease in the infection and expression of CD95 ready for spontaneous apoptosis of lymphocytes in vitro is a negative sign since it is associated with switching to a less efficient viral infection in humoral immune protection path and inherent switching displays such immunosuppression , especially in the form of suppression of the proliferative response of T lymphocytes to PHA [ 25].

Thus, this work is to study the state of apoptosis of immune cells in acute EBV infection is relevant and requires further study.

Therefore:

1. In children with acute Epstein-Barr virus infection has been a violation of the expression of markers of apoptosis of immune cells.
2. In children, the level of Fas/Apo-1 exceeded the reference value of 2,9 times the level of Bcl-2 was increased by 2,3 times, the level of Bax – 3,.2 times the level INFγ - up to 3,1 times the level TNFα - 3 times the level of annexin V (Ann V) – 2,5 times as compared with those in the comparison group.