45 children aged 3–15 years were examined. The aim of the study was to determine the readiness for apoptosis of peripheral blood lymphocytes and neutrophils by determining apoptosis markers - CD95, CD95L, BSL2. When assessing apoptosis of immunocompetent cells, a decrease in the readiness for programmed cell death of lymphocytes and an increase in neutrophilic granulocytes were found. Most pronounced changes registered in age group 7–15 years with a disease experience of more than 3 years. The data obtained may be a sign of suppression of the programmed death of autoreactive lymphocytes in the pancreatic tissue, which contributes to the prolongation of the immune response. An increase in the proportion of leukocyte cells expressing CD95L may enhance the processes of programmed cell death in pancreatic islet β-cells infiltrated with immunocompetent cells.
neutrophil apoptosis
apoptosis of lymphocytes
type 1 diabetes
1. Pekareva E. V. Markers of apoptosis in patients diabetes Type 1 at the onset of the disease / E. V. Pekareva et al. // Diabetes mellitus. - 2009. - No. 4. - S. 86-89.
2. Pekareva E. V. The role of apoptosis in the pathogenesis of type 1 diabetes mellitus / E. V. Pekareva, T. V. Nikonova, O. M. Smirnova // Diabetes mellitus. - 2010. - No. 1. - P.45-48.
3. Adeghate E. An update on the etiology and epidemiology of diabetes mellitus / E. Adeghate, P. Schattner, E. Dunn // Ann NY. Acad Sci, 2006. - Vol. 1084. – P. 1–29.
4. Clinical significance of neutrophil apoptosis in peripheral blood of patients with type 2 diabetes mellitus / C. Sudo et al. // Lab Hematol. 2007; 13(3):108-12 (reduced).
5. Filep J. G. Neutrophil apoptosis: a target for enhancing the resolution of inflammation / J. G. Filep, E. l. Kebir // J. Cell Biochem. - 2009. - Vol. 108. – P. 1039–1046.
6. Human polymorphonuclear neutrophil responses to Burkholderia pseudomallei in healthy and diabetic subjects / S. Chanchamroen et al. // Infect Immun. - 2009. - Vol. 77. - P. 456-463 (reduced apoptosis).
7. Impact of Lymphocyte Apoptosis in Diabetes mellitus / K. A. Awadhesh et al. // Asian Journal of Medical Sciences. - 2011. - No. 2. - P. 1-6.
8. Inflammation is more persistent in type 1 diabetic mice / D. T. Graves et al. // J. Dent. Res., 2005. Vol. 84. – P. 324–328.
9. Juliana C. Alves Infections in patients with diabetes mellitus: A review of pathogenesis / C. Juliana, C. Janine, C. Alves // Indian J. Endocrinol. Metab. – March 2012. – Supp l1. – No. 16. – P. 27–36.
10. Luo H. R. Constitutive neutrophil apoptosis: mechanisms and regulation / H. R Luo, F. Loison // Am. J. Hematol. - 2008. - Vol. 83. – P. 288–295.
Introduction
Type 1 diabetes mellitus (DM1) is a polygenic, multifactorial disease associated with the formation of autoantibodies and autoreactive T-lymphocytes to pancreatic β-cells.
The leading links in the pathogenesis of autoimmune lesions are immune dysregulation and programmed cell death.
Controlled apoptosis is considered today as the main mechanism for maintaining the optimal balance of cells in the focus of inflammation, limiting the expansion of activated clones and preventing the development of autoimmune reactions. If a defect occurs in its implementation, activated immune cells can accumulate, leading to autoimmune diseases.
Purpose of the study: study of activation markers of CD95, CD95L, Bsl2 apoptosis on peripheral blood lymphocytes and neutrophils in type 1 diabetes mellitus in children.
Material and research methods
45 children with type 1 diabetes mellitus aged 3-15 years were examined. Group I included 20 children aged 3-6 years (preschoolers), group II - 12 children aged 7-15 years (schoolchildren) with a disease duration of less than 3 years, group III - 13 children aged 7-15 years (schoolchildren) with the experience of the disease for more than 3 years. The control group consisted of 30 healthy children aged 3-6 (15) and 7-15 (15) years old. The study was conducted on the basis of the endocrinological department of the Children's City Clinical Hospital named after. G. K. Filippsky, Stavropol.
To assess programmed cell death, the number of lymphocytes and neutrophilic granulocytes expressing apoptosis markers was determined. Lymphocytes were isolated on a Ficoll-Paque density gradient, neutrophils were isolated on a Ficoll-Paque double density gradient and ficoll-urografin (GE Healthcare, Sweden). The cell suspension was washed three times in RPMI-1640 medium (Vector-Best, Russia). In cultures of lymphocytes and neutrophils, the number of cells expressing CD95, CD95L, Bsl2 was estimated by flow cytometry using monoclonal antibodies (Invitrogen, USA).
For statistical analysis data, the software package "Primer of Biostat 4.0", Attestat 10.5.1. was used. To assess intergroup differences, repeated measures analysis of variance was used with the calculation of the Newman-Keuls, Dunn criteria.
Quantitative values were characterized by an abnormal distribution and were presented as a median and interquantile (25th and 75th percentile) range (Me (Q1-Q)). Differences at p<0,05.
Results and its discussion
The study found a decrease in the number of lymphocytes expressing Fas receptors (CD95) in patients of all groups compared with healthy children (Table 1). The minimum indicators were noted in children aged 7-15 years with a disease experience of more than 3 years (Table 1).
Table 1
Indicators of apoptosis of lymphocytes in children with type 1 diabetes mellitus
|
Clinical groups |
||||
|
3-6 years old |
DM1 (I) (n=20) |
17,7(15,9-19,43) * ** |
7,4(5,81- 8,94) * ** |
70,2(68,56-71,76) * ** |
|
Control group |
28,0(26,08-30,0) |
9,2(8,04- 10,25) |
65,9(62,82-69,05) |
|
|
7-15 years old |
20,5(17,94-23,02) * ** |
11,6(10,12-13,14) * ** |
70,3(65,72-74,9) * ** |
|
|
13,9(10,04-17,73) * ** |
15,6(14,26-16,87) * ** |
79,5(75,47-83,59) * ** |
||
|
Control group |
26,5 (24,20-28,84) |
8,14 (6,49-9,78) |
60,3(56,97-63,66) |
*- p<0,05 - по сравнению с контрольной группой, **- p<0,05 - по сравнению с группой
When assessing the level of expression of anti-apoptotic markers (Bsl2), its increase was revealed on lymphocytes of children of all groups, more pronounced in schoolchildren with a disease duration of more than 3 years, which also indicates a violation of Fas-dependent apoptosis in children with type 1 diabetes mellitus, leading to slowing down the processes of cell death of autoreactive forms of lymphocytes.
Our results may be an indirect sign of the suppression of the programmed death of activated lymphocytes in the pancreatic tissue, which contributes to the prolongation of the immune response.
The level of apoptotic readiness of lymphoid cells depends on the duration of the disease and decreases in children with DM1 experience of more than 3 years.
Previously, it was shown that lymphocyte resistance to apoptosis is detected in diabetes mellitus, which may explain the nature and duration of the autoimmune response.
In the culture of lymphocytes from children with diabetes mellitus, an increase in the percentage of lymphocytes expressing CD95L was found (Table 1) compared with the group of healthy children. The highest rates were determined in children aged 7-15 years with a disease experience of more than 3 years (Table 1).
It is known that in DM1 pancreatic islets are infiltrated with immune cells producing a wide range of cytokines, which is accompanied by aberrant expression of membrane receptors. Under the influence of an increased concentration of glucose and cytokines, β-cells begin to express CD95 on their surface, which is practically absent in the norm.
An increase in CD95L expression on lymphoid cells probably causes a more pronounced apoptotic process in pancreatic β-cells and their subsequent removal.
In recent years, it has been shown that neutrophilic granulocytes are actively involved in the formation of autoimmune inflammation. The reaction of neutrophils aimed at the localization and elimination of autoantigens largely depends on the strength and duration of the antigenic effect on the immune system, as well as the initial level of functional activity of cells.
We found that the course of diabetes mellitus in children is accompanied by an increase in the percentage of neutrophils expressing apoptosis markers (CD95) and a decrease in the proportion of cells that have Bsl2 antiapoptotic proteins on their surface (Table 2).
table 2
Indicators of neutrophil apoptosis in children with type 1 diabetes mellitus
|
clinical groups |
||||
|
3-6 years old |
DM1 (I) (n=20) |
75,1(71,49-78,72) * ** |
9,5 (8,63- 10,32) * ** |
3,68 (3,46-3,90 * ** |
|
control group |
59,2 (56,31- 62,01) |
7,35 (6,58- 8,12) |
||
|
7-15 years old |
DM1, disease experience less than 3 years (II) (n=12) |
77,6(71,15-83,99) * ** |
9,5(8,14-10,92) * ** |
3,99(2,9- 5,08) * ** |
|
DM1, disease experience more than 3 years (III) (n=13) |
87,9(84,24-91,63) * ** |
12,1(10,22-13,96) * ** |
2,78(2,36-3,19) * ** |
|
|
control group |
58,43(54,95- 1,90) |
*- p<0,05 - по сравнению с контрольной группой, **- p<0,05 - по сравнению с группой III (Newman-Keuls criterion, Dunn criterion).
With a comparative intergroup characteristic, the maximum indicators of CD95 (p<0,05) и минимальные Bsl2 (p<0,05) отмечены у детей 7-15 лет с длительностью заболевания более 3-х лет.
An increase in the percentage of polymorphonuclear leukocytes with CD95L on their surface was revealed. The highest rates were noted in children of schoolchildren with the experience of the disease for more than 3 years.
The results of the study of PMNL apoptosis in diabetes mellitus presented in the literature are controversial. There is evidence of an increase in the rate of apoptosis of peripheral blood neutrophils in DM1 and DM2.
However, a number of studies have found a decrease in apoptosis of neutrophilic granulocytes in patients with type 1 diabetes, especially under conditions of hyperglycemia, which probably initiates chronic inflammation processes with tissue damage, and also predisposes to protracted bacterial infections in patients with type 1 diabetes mellitus.
Our results suggest that patients with DM1 have an increased predisposition of PMNL to apoptosis, which may be a manifestation of a protective reaction aimed at eliminating the “surplus” of active neutrophils, the formation of which enhances tissue damage.
An increase in the apoptotic potential of neutrophilic granulocytes is a reflection of the active involvement of PMNL in the immunopathogenesis of the disease.
An increase in CD95L expression on neutrophilic granulocytes in patients with diabetes mellitus can probably contribute to the elimination of not only pancreatic cells, but also their own leukocyte cells.
Thus, when evaluating apoptosis of immunocompetent cells in children with type 1 diabetes, a decrease in the readiness for programmed cell death of lymphocytes and an increase in polymorphonuclear leukocytes were found.
The most pronounced changes are recorded in the age group of 7-15 years with a disease experience of more than 3 years. In children of all groups, an increase in the proportion of leukocyte cells expressing CD95L on their surface was revealed.
It is known that PMNL are a link between innate and adaptive immunity and play a leading role in antibacterial protection.
An increase in their apoptotic activity can cause a child's low age resistance and susceptibility to infectious diseases.
A decrease in the number of lymphoid cells sensitive to the induction of apoptosis is an indirect sign of the suppression of programmed cell death and impaired elimination of activated forms of lymphocytes.
conclusions
1. In children with type 1 diabetes, there is a decrease in the readiness for apoptosis of peripheral blood lymphocytes, an increase in neutrophilic granulocytes, which is accompanied by a change in the expression of CD95 and Bsl2 and depends on the duration of the disease.
2. An increase in CD95L expression on lymphocytes and neutrophilic granulocytes in DM1 may enhance the processes of programmed cell death in pancreatic islet β-cells infiltrated with immunocompetent cells.
Reviewers:
Shchetinin E.V., Doctor of Medical Sciences, Professor, Vice-Rector for Research and Innovation, SSMU, Head of the Department of HBO HPE "Stavropol State Medical University" of the Ministry of Health of the Russian Federation, Stavropol.
Golubeva M.V., Doctor of Medical Sciences, Professor, Head of the Department of Pediatric Infectious Diseases, Stavropol State Medical University, Stavropol State Medical University, Stavropol.
Bibliographic link
Barycheva L.Yu., Erdni-Goryaeva N.E. MARKERS OF APOPTOSIS OF IMMUNOCOMPETENT CELLS IN TYPE 1 DIABETES MELLITUS IN CHILDREN // Modern problems of science and education. - 2013. - No. 4.;URL: http://science-education.ru/ru/article/view?id=9953 (date of access: 07/18/2019). We bring to your attention the journals published by the publishing house "Academy of Natural History" 1
Markova A.A. oneKashkina E.I. one Rubtsov V.S. 1 Lyakisheva R.V. one
1 Saratov State Medical University named after V.I. IN AND. Razumovsky, Saratov
The expression of apoptosis and proliferation markers in 61 patients with ulcerative colitis was analyzed depending on the duration, severity of the disease, and the location of the inflammatory process in the colon. The comparison group consisted of 15 practically healthy people. Examination of patients was carried out using clinical, laboratory, endoscopic, morphological methods. In biopsy samples of the colon mucosa, the expression of immunohistochemical markers Ki-67, P53, BAX, and CEA was determined. The study revealed a statistically significant decrease in the proliferative activity of the colon mucosa in patients with ulcerative colitis compared with a group of healthy individuals, as well as a decrease in proliferation processes and an increase in the expression of apoptosis markers as the duration and severity of clinical manifestations of the disease increase.
nonspecific ulcerative colitis
markers of apoptosis and proliferation
1. Aruin L.I., Kapuller L.L., Isakov V.A. Morphological diagnosis of diseases of the stomach and intestines. - M.: Triada-X, 1998. - 496 p.
2. Gastroenterology and hepatology: diagnosis and treatment: a guide for doctors / ed. A.V. Kalinina, A.F. Loginova, A.I. Khazanov. - 2nd ed., revised. and additional – M.: MEDpress-inform, 2011. – 864 p.: ill.
3. Brown D. Gatter K. Monoclonal antibody Ki-67: its use in histopathology // Histopathology. - 1990. - No. 17. -
4. Bruno S., Darynkiewich Z. Cell cycle dependent expression and stability of the nuclear protein detected by Ki-67 antibody in HL-60 cells, Cell. Profile. - 1992. - No. 25. -
5. A comparison of proliferation markers (BrdUrd, Ki-67, PCNA) determined at each cell position in the crypts of normal human colonic mucosa / M. Bromley, D. Rew, A. Becciolini
et al. // EUR. J. Histochem. - 1996. - Vol. 40. – P. 89–100.
6. Holt P.R., Moss S.F., Kapetanakis A.M. Is Ki-67 a better proliferative marker in the colon then proliferating cell nuclear antigen? cancer. Epidimiol // Biomarkers. Prev. - 1997. -
No. 6. - P. 131-135.
7. McCormick D., Chong C., Hobbs C. et al. Detection of the Ki-67 antigen in fixed and wax-embedded section with the monoclonal antibody MIB-1 // Histopathology. - 1993. -
No. 22. - P. 355-360.
8. Reed J. C. Bcl-2 and regulation of programmed cell death // J. Cell. Biol. - 1994. - Vol. 124. – P. 1–6.
Nonspecific ulcerative colitis (NUC) is one of the most severe diseases of the gastrointestinal tract, leading to disability and death of patients.
In recent decades, there has been an increase in the incidence of UC in various countries of the world, which is largely due to the improvement in the diagnosis of this pathological process.
Currently, in the diagnosis of UC, an integrated approach is used using X-ray, endoscopic, and histological research methods. One of the promising methods for assessing the condition of the colon mucosa is immunohistochemistry with the determination of markers of proliferation and apoptosis.
Among the methods of immunohistochemical studies, the apoptosis markers bcl and p53 have found the widest application. It is known that proteins of the bcl family are either inducers of apoptosis (Bad, Bax, Bak, etc.) or inhibitors of apoptosis (Bcl-2, Bcl-XL, BOO, etc.). It should be noted that the p53 protein is detected in many transformed cells. Its functions are aimed at preventing the transfer of damaged genetic information from one generation of cells to another, including through the initiation of apoptosis. A high content of p53 leads to an increase in the concentration of Bax in the cell and a decrease in the concentration of bcl-2, which contributes to cell death by apoptosis.
Several antigens are used as proliferation markers. Proliferating Cell Nuclear Antigen (PCNA) is involved not only in cell proliferation, but also in DNA repair after damage, which makes this antigen conditionally specific to the cell cycle, since DNA repair can be carried out in the resting phase. Another antigen significantly associated with cell cycle phases is Ki-67. The expression of this protein occurs during the presynthetic phase, increases during the cell cycle, and decreases sharply during the mitotic phase. This protein, unlike PCNA, is not involved in DNA repair. Expression of Ki-67 makes it possible to identify cells that are in all phases of the cell cycle, except for the resting phase.
The aim of the study was to analyze the expression of apoptosis and proliferation markers in patients with ulcerative colitis, depending on the duration, severity of the disease, and the localization of the inflammatory process.
Materials and methods of research
The main group consisted of 61 patients with UC aged 19 to 66 years (27 women and 34 men), the comparison group consisted of 15 practically healthy people. Examination of patients was carried out using clinical, laboratory, endoscopic, morphological methods, as well as with the help of immunohistochemical studies of biopsy specimens of the colon mucosa.
Patients with UC were divided into groups depending on the severity of clinical manifestations, the duration of the disease, and the localization of the inflammatory process.
The proliferative activity of cells was determined by the proliferative indicator Ki-67 according to the formula:
where X is the number of nuclei in the field of view of the microscope. The counting was carried out in at least 10 fields of view.
Apoptosis was judged by the expression of p53 and BAX proteins in the superficial and glandular epithelium of the large intestine. To assess regenerative processes in the colon mucosa in UC, the expression of cancer embryonic antigen (CEA) was determined.
Research results and discussion
An immunohistochemical study determined the dependence of the expression of these markers on the duration of UC, the severity of its clinical manifestations, and the prevalence of the inflammatory process in the colon.
During statistical processing of the obtained data after testing for equality of variances and normality of distribution, it was proved that the sample does not correspond to the law of normal distribution, therefore, nonparametric criteria were used to compare groups. The median, upper and lower quartiles were used to describe quantitative characteristics.
Thus, in healthy individuals, the Ki-67 PI was 54 (46; 67), which indicates a high proliferative activity of colon cells (table).
Proliferation index Ki-67 of epithelial cells of the colon mucosa (%) in healthy people and patients with ulcerative colitis, depending on its duration, severity and localization
|
Analyzed groups |
Ki-67 values |
Reliability of differences |
|
|
Comparison group |
|||
|
Disease duration |
p 0 ≤ 0.001 R 1 ≤ 0,02 p 0 ≤ 0.001 R 1 ≥ 0,05 |
||
|
The severity of the current medium-heavy |
p 0 ≤ 0.004 R 2 ≤ 0,05 p 0 ≤ 0.004 R 2 ≤ 0,02 |
||
|
Localization of the lesion distal left-sided total |
p 0 ≤ 0.002 R 3 ≥ 0,05 p 0 ≤ 0.002 R 3 ≥ 0,05 |
Notes:
- R 0 - significance of differences with the control group;
- R 1 - significance of differences with disease duration less than 1 year;
- R 2 - reliability of differences with a mild course of the disease;
- R 3 - reliability of differences with the distal localization of the disease.
BAX expression in healthy individuals was absent in 80% of cases, and low expression of the marker was noted in 20%. CEA expression was observed in 50% of cases and was also low.
All patients, depending on the duration of the disease, were divided into 3 groups: the 1st with UC lasting up to 1 year, the 2nd with a duration of 1 to 5 years, and the 3rd - more than 5 years.
As follows from the table, the increase in Ki-67 PI in the group of patients with a disease duration of 1 to 5 years compared with group 1 ( R≤ 0.02) can be explained by increased proliferative activity of cells, reflecting reparative processes in the colon mucosa. Low PI Ki-67 in the group of patients with ulcerative colitis lasting up to 1 year, amounting to 31 (25; 36), may indicate a pronounced decrease in proliferative processes in the intestinal mucosa at the onset of the disease.
When studying the p53 index depending on the duration of the disease, no regularities in the expression of this protein were revealed, however, there is a difference in the expression of this marker between a group of healthy individuals and a group of patients with a disease duration of 1 to 5 years ( R≤ 0.05) and more than 5 years ( R ≤ 0,03).
BAX expression was determined in both the superficial and glandular epithelium of the colon. It was found that the expression of this marker in the surface epithelium and in the epithelium of the glands is almost the same in each individual case. BAX expression in UC patients was detected in 100% of cases, statistically significant differences in the expression of the marker were found between the control group and UC patients ( R ≤ 0,01).
When comparing the intensity of CEA expression, a significant increase was noted with an increase in the duration of the disease. In the comparison group, CEA expression was manifested only in isolated cases ( R ≤ 0,003).
Depending on the severity of clinical manifestations of UC, a change in Ki-67 PI was also detected (see table). Ki-67 PI decreased in patients with moderate disease ( R≤ 0.05) and severe form ( R≤ 0.02) compared with patients with mild course, there was also a difference between the group of healthy individuals and patients with UC of any severity ( R ≤ 0,004).
The change in the expression of the p53 marker depended on the severity of the exacerbation (12.5% for mild course; 35.7% for moderate severity and 50% for severe). Statistically significant differences were obtained between groups of healthy individuals and patients with UC of moderate and severe forms of the disease ( R ≤ 0,03).
BAX and CEA were expressed in 100% of cases, however, no dependence of the expression intensity of these markers on the severity of clinical manifestations of UC was found. Differences in the expression of markers were established between the comparison group and patients with UC, regardless of the severity of clinical manifestations (p≤0.01).
When analyzing the dependence of Ki-67 IP on the localization of the inflammatory process, a significant increase in the expression of the marker in the group of patients with UC compared with healthy people was revealed ( R≤ 0.002), however, in a comparative analysis within the main group, no statistically significant differences between marker expression and different localization of the process were obtained ( R ≥ 0,05).
Expression of p53 was not detected in all patients. In patients with distal colitis, a positive result was obtained only in 20% of cases, with left-sided colitis in 18% of patients. With total intestinal damage, p53 expression was detected in 100% of cases ( R≤ 0.03 compared to distal colitis). It should be noted that expression was most intense in areas with signs of epithelial metaplasia.
The intensity of BAX expression in the group with total colitis was significantly higher than in the group with distal colitis ( R ≤ 0,03).
CEA expression did not depend on the localization of the inflammatory process, however, it was significantly higher in patients with UC compared with the comparison group ( R ≤ 0,03).
In nonspecific ulcerative colitis, the proliferative activity of colonic mucosal epithelial cells is significantly lower, and apoptosis rates are higher than in the absence of its inflammatory changes.
An increase in the duration of nonspecific ulcerative colitis is accompanied by a low proliferative activity of the colonic mucosal epithelium, which can impair its repair.
With an increase in the severity of clinical manifestations of nonspecific ulcerative colitis, not only a decrease in the degree of proliferative activity of colon epithelial cells is noted, but also an increase in apoptosis activation indicators.
With the spread of the inflammatory process to the proximal parts of the colon, an increase in apoptosis was revealed.
Reviewers:
Shvarts Yu.G., Doctor of Medical Sciences, Professor, Head. Department of Faculty Therapy, Saratov State Medical University named after V.I. Razumovsky" of the Ministry of Health and Social Development of Russia, Saratov;
Fedorina T.A., Doctor of Medical Sciences, Professor, Head. Department of General and Clinical Pathology: Pathological Anatomy, Pathological Physiology, Samara State Medical University, Ministry of Health and Social Development of Russia, Samara.
The work was received by the editors on 09.12.2011.
Bibliographic link
Markova A.A., Kashkina E.I., Rubtsov V.S., Lyakisheva R.V. PECULIARITIES OF EXPRESSION OF MARKERS OF APOPTOSIS AND PROLIFERATION IN PATIENTS WITH NON-SPECIFIC ULCERATIVE COLITIS // Fundamental Research. - 2012. - No. 2. - P. 79-82;URL: http://fundamental-research.ru/ru/article/view?id=29400 (date of access: 07/18/2019). We bring to your attention the journals published by the publishing house "Academy of Natural History"
CAD (caspase activated DNase) into fragments in multiples of 180-200 nucleotides. Apoptosis results in the formation of apoptotic bodies - membrane vesicles containing integral organelles and fragments of nuclear chromatin. These bodies are taken up by neighboring cells or macrophages through phagocytosis. Since the extracellular matrix is not affected by cellular enzymes, even with a large number of apoptotic cells, inflammation is not observed.
The process of apoptosis is necessary for the physiological regulation of the number of cells in the body, for the destruction of old cells, for the formation of lymphocytes that are not reactive to their antigens (self-antigens), for the autumn leaf fall of plants, for the cytotoxic action of T-killer lymphocytes, for the embryonic development of the organism ( disappearance of skin membranes between fingers in bird embryos) and others.
Violation of normal cell apoptosis leads to uncontrolled cell proliferation and the appearance of a tumor.
1. Significance of apoptosis
Apoptosis is an integral part of the vital activity of most multicellular organisms. It plays a particularly important role in development processes. For example, the limbs of tetrapods are laid as spade-shaped grow, and the formation of fingers occurs due to the death of cells between them. Cells no longer needed are also subject to apoptosis, thus the tail in tadpoles is destroyed in particular during metamorphosis. In the nervous tissue of vertebrates during embryonic development, more than half of the neurons die by apoptosis immediately after formation.
Also, apoptosis is part of the control system for the "quality" of cells, it allows you to destroy those that are incorrectly located, damaged, non-functional or potentially dangerous to the body. An example is B-lymphocytes, which die if they do not carry useful antigen-specific receptors or are auto-reactive. By apoptosis, most of the lymphocytes that are activated during infection also die after it has been overcome.
In adult organisms, the simultaneous regulation of cell proliferation and apoptosis makes it possible to maintain the size of the whole individual and its individual organs. For example, after implantation of the drug phenobarbital, which stimulates the proliferation of hepatocytes, the liver increases in rats. However, immediately after the cessation of the action of this substance, all excess cells undergo apoptosis, resulting in the size of the liver returning to normal.
Apoptosis also occurs when a cell "feels" a large amount of internal damage that it cannot repair. For example, in the event of DNA damage, a cell can transform into a cancer cell, so that this does not happen, it, under normal conditions, "commits suicide." Also, a large number of cells infected with viruses die by apoptosis.
2. Markers of apoptotic cells
Apoptosis markers
Detection of DNA fragmentation in apoptotic cells by TUNEL method Preparation of mouse liver tissue, apoptotic cell nucleus has a brown color, optical microscopy.
Detection of DNA fragmentation in apoptotic cells by agarose gel electrophoresis. Left: DNA isolated from apoptotic cells - "DNA ladder" is visible; middle: markers; case: DNA control sample from untreated cells. Cell line H4IIE (rat hepatoma), apoptosis inducer - paraquat, visualization with etidium bromide.
Top: Detection of chromatin condensation and fragmentation by staining with fluorescent dye (Hoechst 34580). Middle: Detection of translocation of phosphadidylserine to the outer leaflet of the plasmalemma by staining with annexin V. Bottom: Bright field micrograph of apoptotic cells. Cell line - Jurkat, apoptosis inducer - TRAIL, confocal and light saw optical microscopy.
Cells that die by apoptosis can be recognized by a number of morphological features. They become smaller and denser (pyknosis), round and lose pseudopodia, the cytoskeleton collapses in them, the nuclear membrane disintegrates, the chromatin condenses and fragments. A large number of vesicles appear on the surface of the cells, if the cells are large enough, then they disintegrate into fragments surrounded by membranes - apoptotic bodies.
In apoptotic cells, in addition to morphological changes, a large number of biochemical changes also occur. In particular, DNA is cut by special nucleases in the linker regions between nucleosomes into fragments of equal length. Therefore, when separating the entire DNA of an apoptotic cell using electrophoresis, a characteristic "ladder" can be observed. Another method for detecting DNA fragmentation is to mark its free ends using the TUNEL method ( T erminal deoxynucleotidyl transferase d U TP n ick e nd l abeling ) .
The plasma membrane of apoptotic cells also undergoes changes. Under normal conditions, the negatively charged phospholipid phosphatidylserine is contained only in its inner (returned to the cytosol) layer, but during apoptosis it "jumps" into the outer leaflet. This molecule serves as the "eat me" signal to nearby phagocytes. Phosphatidylserine-induced uptake of apoptotic cells, unlike other types of phagocytosis, does not result in the release of inflammatory mediators. The described change in the plasma membrane underlies another method for detecting cells that die by apoptosis - staining with anexin V, which specifically binds to phosphatidylserine.
3. Caspase - mediators of apoptosis
Cellular systems that ensure the passage of apoptosis are similar in all animals; the caspase family of proteins occupies a central place in them. Caspases are proteases that have a cysteine residue in their active site and cut their substrates at a specific aspartic acid residue (hence the name: c from cysteine and asp from aspartic acid). Caspases are synthesized in the cell in the form of inactive procaspases, which can become substrates for other already activated caspases, which cut them in one or two places at the aspartate residue. Two formed fragments - a larger and a smaller one - are interconnected, forming a dimer that associates with the same dimmer. The tetramer formed in this way is an active protease, which can cut substrate proteins. In addition to regions corresponding to the larger and smaller subunits, procaspases sometimes also contain inhibitory prodomains that are degraded after cleavage.
As a result of cleavage and activation of some caspases by others, a protealytic cascade is formed, which significantly enhances the signal and makes apoptosis an irreversible process from a certain moment. Those procaspases that start this cascade are called initiatory ones, and their substrates are called effector ones. After activation, effector caspases can cleave other effector procaspases or target proteins. The targets of effector caspases that are destroyed during apoptosis include, in particular, nuclear lamina proteins, the splitting of which leads to the breakdown of this structure. It also degrades the protein, under normal conditions inhibits CAD endonucleases, as a result of which DNA fragmentation begins. Caspase and cytoskeletal and intercellular adhesion proteins are cleaved, as a result of which apoptotic cells round and detach from neighboring cells, and thus become easier targets for phagocytes.
The set of caspases required for apoptosis to proceed depends on the type of tissue and the pathway by which cell death is activated. For example, in mice, when the gene encoding effector caspases-3 is "turned off", apoptosis does not occur in the brain, but normally proceeds in other tissues.
Procaspase genes are active in healthy cells, and therefore proteins are necessary for the occurrence of apoptosis and are constantly present, only their activation is needed to trigger cell suicide. The initiator procaspases include a long prodomain containing CARD ( caspase recruitment domain , caspase attraction domain). CARD allows procaspase initiators to attach to adapter proteins to form activation complexes when the cell receives a signal that stimulates apoptosis. In activation complexes, several pro-caspase molecules are in close proximity to each other, which is enough for them to enter the active state, after which they cut each other.
The two best understood signaling pathways for activation of the caspase cascade in mammalian cells are called extrinsic and intrinsic (mitochondrial), each using its own initiator procaspase.
4. Ways of activation of apoptosis
4.1. outer path
The cell can receive a signal inducing apoptosis from outside, for example, from cytotoxic lymphocytes. In this case, the so-called external path is activated ( extrinsic pathway) Starting with death receptors. Death receptors are transmembrane proteins belonging to the tumor necrosis factor (TNF) receptor family, such as the TNF receptor itself and the Fas death receptor. They form homotrimers, in which each monomer has an extracellular ligand-binding domain, a transmembrane domain, and a cytoplasmic death domain, attracts and activates procaspases via adapter proteins.
Death receptor ligands are also homotrimerams. They are related to each other and belong to the tumor necrosis factor signaling molecule family. For example, cytotoxic lymphocytes carry Fas ligands on their surface, which can bind to Fas death receptors on the plasmalemma of target cells. In this case, the intracellular domains of these receptors are connected to the adapter protein ( FADD, Fas-associated death domain ), and they, in turn, attract pro-caspase 8 and/or 10 with initiation. As a result of this series of events, a death-inducing signaling complex is formed - DISC ( death inducing signaling complex ). Upon activation in this complex by initiator caspases, they cleave effector procaspases and trigger the apoptotic cascade.
Many cells synthesize molecules that, to a certain extent, protect them from activation of the external pathway of apoptosis. An example of such protection would be the expression of so-called decoy receptors ( decoy receptors), which have extracellular ligand binding domains, but not cytoplasmic death domains, and therefore cannot trigger apoptosis and compete with conventional death receptors for ligands. Cells can also produce proteins that block the extrinsic pathway of apoptosis, such as FLIP, which is similar in structure to procaspases 8 and 10 but has no proteolytic activity. It inhibits the binding of initiator procaspases to the DISC complex.
4.2. Inner path
Apoptosome
Apoptosis can also be triggered from within the cell, such as in the event of cell injury, DNA damage, lack of oxygen, nutrients, or extracellular survival signals. In vertebrates, this signaling pathway is called intrinsic ( intrinsic pathway) Or mitochondrial, the key event in it is the release of certain molecules from the intermembrane space of mitochondria. Cychrome c lies before such zocrema molecules, which enters the electron-transport lance of mitochondria, prote in the cytoplasm performs another function - it comes to the adapter protein Apaf ( apoptotic protease actiuating factor l ), causing it to oligomerize into a wheel-shaped seven-membered structure called the apoptosome. The apoptosome recruits and activates the initiator procaspase-9, which can then activate the initiator procaspase.
In some cells, the extrinsic apoptosis pathway must activate the intrinsic one in order to effectively destroy the cell. The internal pathway is highly regulated by the Bcl-2 family proteins.
4.2.1. Regulation of the intrinsic pathway by Bcl-2 family proteins
The Bcl-2 family includes evolutionarily conserved proteins whose main function is to regulate the release of cytochrome c and other molecules from the intermembrane space of mitochondria. Among them are pro-apoptotic and anti-apoptotic molecules that can interact with each other in various combinations, suppressing each other, the balance between their activity and determining the fate of the cell.
About 20 proteins from this family are now known, all of which contain at least one of the four alpha helical Bcl2 homology domains called BH1-4 ( bcl2 homology). Anti-apoptotic proteins of the Bcl2 family contain all four domains, including Bcl-2 itself, as well as Bcl-X L, Bcl-w, Mcl-1 and A1. Pro-apoptotic proteins are divided into two groups, members of the first of which contain three BH-domains (BH1-3), these are in particular Bak, Bax and Bok (the latter is expressed only in the tissues of the reproductive organs). The most numerous among the Bcl-2 family is the second group of proapoptotic proteins that contain only the BH3 domain (BH3-only), it includes Bim, Bid, Bad, Bik/Nbk, Bmf, Nix/BNIP3, Hrk, Noxa, Puma.
Under normal conditions (i.e., when the cell is not undergoing apoptosis), anti-apoptotic proteins such as Bcl-2 and Bcl-XL bind to pro-apoptotic BH123 proteins (Bax and Bak) and prevent them from polymerizing in the outer mitochondrial membrane to form pores. As a result of the action of a certain apoptotic stimulus, proapoptotic proteins containing only the BH3 domain are activated or begin to be synthesized in the cell. They, in turn, inhibit anti-apoptotic proteins, removing the inhibitory effect on Bak and Bax, or interact directly with the latter and promote their oligomerization and pore formation. Due to the permeabilization of the outer membrane, cytochrome c enters the cytosol, as well as other mediators of apoptosis, such as AIF. apoptosis inducing factor ).
For example, when there is a lack of survival signals in the cell, MAP kinase JNK activates the expression of the BH3 protein Bim, which triggers the internal pathway of apoptosis. In the event of DNA damage, the tumor suppressor p53 accumulates, which stimulates the transcription of genes encoding the BH3 proteins Puma and Noxa, which also ensure the passage of apoptosis. Another BH3 protein, Bid, provides a link between the extrinsic and intrinsic pathways of apoptosis. After activation of death receptors and, as a result, caspase-8, the latter cleaves Bid to form a truncated form of tBid (truncated Bid), which moves to the mitochonria, where it suppresses Bcl-2.
Cell division as a whole is a rather monotonous process called the cell cycle. There are a large number of "checkpoints" in it, which control the transition of the cell from one phase of the cycle to another. Destruction of one or more "checkpoints" can lead to both uncontrolled proliferation and cell death, in particular, to apoptosis. The morphological picture of apoptosis with all the characteristic features (chromatolysis, absence of an inflammatory response, cell cannibalism, etc.) was described by L. Graper and called "physiological cell elimination". In 1971, J. Kerr proposed the term "apoptosis" (from Latin aro - with, ptosis - to fall) by analogy with leaves falling from a tree here and there. During apoptosis, three phases are distinguished - early (cell size reduction, DNA fragmentation into large fragments), intermediate (further DNA fragmentation) and late (apoptotic bodies). Apoptosis plays an important role in the development of the human placenta. With the course of pregnancy, there is an increase in apoptotic changes in a normally functioning placenta.
Tertemiz et al.
in their work showed that apoptosis is involved in the mechanisms of physiological regulation of placental vasculogenesis. Vasculogenesis of the placenta begins on the 21st day of pregnancy and includes the appearance of hemangioblasts and angiogenic cell islands. Placental vasculogenesis was studied using histological (preparations stained with hematoxylin and eosin), immunohistochemical (detection of CD31), molecular genetic (CD31-TUNEL - TdT-mediated X-dUTP nick end labeling) methods and transmission electron microscopy. The study showed that angiogenic cell islands lack CD31-positive cells. However, in cells of primitive capillaries and in a number of stromal cells located between vasculogenic areas, CD31 expression was detected. Morphological examination of preparations stained with hematoxylin and eosin revealed signs of apoptosis in these cells - karyopyknosis and apoptotic bodies. The severity of apoptosis and vasculogenesis in the placenta was directly proportional.
The level of apoptosis is increased in pregnancy disorders such as early termination of pregnancy, ectopic pregnancy, preeclampsia.
Proliferation and differentiation of the cytotrophoblast and development of vessels in the stroma of the villi require an adequate supply of oxygen and nutrients from the intervillous space. Among pregnancy complications, intrauterine growth retardation is one of the leading causes of perinatal mortality. Dysregulation of apoptosis leads to a decrease in the number of syncytiotrophoblast cells, which leads to a decrease in the supply of nutrients to the fetus and to a delay in intrauterine development of the fetus. Levy et al. associate intrauterine growth retardation with preeclampsia and smoking - conditions that lead to oxygen starvation of the placental tissue. S. Y. Dai et al. The placentas of women without bad habits and preeclampsia, but who had intrauterine growth retardation, were studied. The authors suggested that apoptotic changes in placental cells under conditions of oxygen starvation can be regulated by factors activated under hypoxia conditions (hypoxia-inducible factor) - HIF-la, HIF-2a, HIF-1 p.
HIF-1 is the main factor that ensures cell adaptation to hypoxia.
It can alter the expression of a number of genes responsible for erythropoiesis, glycolysis, and angiogenesis. While the HIF-lp heterodimer is detected in all cells of the placenta under any conditions, HIF-la is detected only during hypoxia. Less commonly, under conditions of oxygen starvation, HIF-2a, also known as EPAS-1, is detected in cells. HIF-la and -2a mRNA are found in the placenta throughout pregnancy, but their level varies significantly depending on the gestational age. If the level of HIF-la mRNA remains constant, then the level of HIF-2a mRNA increases with the course of pregnancy. In contrast to HIF-la, HIF-2a is mainly expressed in endothelial cells, playing an important role in angiogenesis and hematopoiesis. In the human placenta, the expression of HIF-la and HIF-2a is maximally expressed in the early stages, which ensures the resistance of cells to the physiological hypoxia that occurs in this period of pregnancy. In addition, increased expression of these factors was noted in preeclampsia.
A consequence of the stimulating effect of these factors on apoptosis is a delay in intrauterine development of the fetus.
So, in the study by S. Y. Dai et al. the apoptotic index in villous syncytiotrophoblast was 1.45±1.26% in the group with intrauterine growth retardation and 0.18±0.16 in the control group, where intrauterine growth retardation was not observed (p
At the same time, macroscopically detectable infarcts were significantly more common in placentas of the group with intrauterine growth retardation (50%) than in the control group (22%). The degree of fibrinoid deposition in the intervillous space was also slightly higher in the group with intrauterine growth retardation. The mitotic activity of trophoblast elements and blood and vascular cells at 6 and 12-14 weeks of gestation were studied by Challier et al. The authors noted at 6 weeks' gestation the presence of mitotic figures and the presence of Kd67-positive nuclei in cytotrophoblast cells and erythroblasts. In the cytotrophoblast of the villi, the number of Ki67-positive nuclei decreased by 12-14 weeks of pregnancy, remaining only in the cell islands of the extravillous cytotrophoblast. In erythroblasts, Ki67 is no longer detected by this period. In endothelial cells at 6 weeks of gestation, mitotic figures and Ki67 expression were absent; at 12–14 weeks of gestation, the UEA1 lectin was detected. The absence of mitotic figures and Ki67 expression in endothelial and perivascular cells at 6 weeks of gestation indicates a direct dependence of vasculogenesis on stromal cells, and to a greater extent than on trophoblast.
The control of the cell cycle of eukaryotic cells is carried out by a family of kinases, in particular cyclin-dependent kinases. From the first trimester of pregnancy in the nuclei of the cytotrophoblast and the endothelium of adjacent vessels, cyclin D1 is detected, the expression of which progressively increases by the third trimester of pregnancy. CDK4 is detected in the cytotrophoblast nuclei both in the first and third trimester of pregnancy, while COC4-positive endothelial cells are observed only at the end of the third trimester. This suggests that the D1/CDK4 complex is involved in the regulation of cytotrophoblast cell proliferation throughout pregnancy and in the control of angiogenesis in the third trimester of pregnancy. Violations of the endocrine, immunological balance, accumulation of free radicals lead to increased apoptosis in placental tissues. Thus, in preeclampsia, cytotrophoblast differentiation and the process of its invasion into the uterus are disrupted, which is largely due to apoptosis. It is known that apoptosis is much more common in the mature placenta during pregnancy complicated by fetal growth retardation, and the p53 protein plays the main role in the regulation of this process, while the Bcl-2 protein does not participate in it. Numerous studies point to overexpression of p53 and, consequently, to an increase in apoptotic cells in the cytotrophoblast in chorionic carcinoma and hydatidiform mole.
