Other

Determination of cytokines. Cytokines

This chapter will consider an integrated approach to assessing the cytokine system using the previously described modern research methods.

First, we will outline the basic concepts of the cytokine system.

Cytokines are currently considered as protein-peptide molecules produced by various cells of the body and carrying out intercellular and intersystem interactions. Cytokines are universal regulators of the cell life cycle; they control the processes of differentiation, proliferation, functional activation and apoptosis of the latter.

Cytokines produced by cells of the immune system are called immunocytokines; they represent a class of soluble peptide mediators of the immune system, necessary for its development, functioning and interaction with other body systems (Kovalchuk L.V. et al., 1999).

As regulatory molecules, cytokines play an important role in the implementation of innate and adaptive immune reactions, ensure their interaction, control hematopoiesis, inflammation, wound healing, the formation of new blood vessels (angiogenesis) and many other vital processes.

Currently, there are several different classifications of cytokines, taking into account their structure, functional activity, origin, and type of cytokine receptors. Traditionally, in accordance with their biological effects, it is customary to distinguish the following groups of cytokines.

1. Interleukins(IL-1-IL-33) - secretory regulatory proteins of the immune system, providing mediator interactions in the immune system and its connection with other systems of the body. Interleukins are divided according to their functional activity into pro- and anti-inflammatory cytokines, lymphocyte growth factors, regulatory cytokines, etc.

3. Tumor necrosis factors (TNF)- cytokines with cytotoxic and regulatory actions: TNFa and lymphotoxins (LT).

4. Hematopoietic cell growth factors- stem cell growth factor (Kit - ligand), IL-3, IL-7, IL-11, erythropoietin, trobopoietin, granulocyte-macrophage colony-stimulating factor - GM-CSF, granulocyte CSF - G-CSF, macrophage-

ny CSF - M-CSF).

5. Chemokines- C, CC, CXC (IL-8), CX3C - regulators of chemotaxis of various cell types.

6. Non-lymphoid cell growth factors- regulators of growth, differentiation and functional activity of cells of various tissues (fibroblast growth factor - FGF, endothelial cell growth factor, epidermal growth factor - EGF of the epidermis) and transforming growth factors (TGFβ, TGFα).

Among others, in recent years, a factor that inhibits the migration of macrophages (migration inhibitory factor - MIF), which is considered as a neurohormone with cytokine and enzyme activity, has been actively studied (Suslov A.P., 2003; Kovalchuk L.V. et al.,

Cytokines differ in structure, biological activity and other properties. However, along with differences, cytokines have general properties, characteristic of this class of bioregulatory molecules.

1. Cytokines are, as a rule, glycosylated polypeptides of medium molecular weight (less than 30 kD).

2. Cytokines are produced by cells of the immune system and other cells (for example, endothelium, fibroblasts, etc.) in response to an activating stimulus (pathogen-associated molecular structures, antigens, cytokines, etc.) and participate in the reactions of innate and adaptive immunity, regulating their strength and duration. Some cytokines are synthesized constitutively.

3. Secretion of cytokines is a short process. Cytokines are not stored as preformed molecules, but their

synthesis always begins with gene transcription. Cells produce cytokines in low concentrations (picograms per milliliter).

4. In most cases, cytokines are produced and act on target cells located in close proximity (short-range action). The main site of action of cytokines is the intercellular synapse.

5. Redundancy The cytokine system is manifested in the fact that each cell type is capable of producing several cytokines, and each cytokine can be secreted by different cells.

6. All cytokines are characterized by pleiotropy, or multifunctionality of action. Thus, the manifestation of signs of inflammation is due to the influence of IL-1, TNFα, IL-6, IL-8. Duplication of functions ensures reliable operation of the cytokine system.

7. The action of cytokines on target cells is mediated by highly specific, high-affinity membrane receptors, which are transmembrane glycoproteins, usually consisting of more than one subunit. The extracellular part of the receptors is responsible for cytokine binding. There are receptors that eliminate excess cytokines in the pathological focus. These are so-called decoy receptors. Soluble receptors are an extracellular domain of a membrane receptor separated by an enzyme. Soluble receptors are able to neutralize cytokines, participate in their transport to the site of inflammation and in their removal from the body.

8. Cytokines work on the network principle. They can act in concert. Many functions initially attributed to one cytokine appear to be due to the coordinated action of several cytokines. (synergy actions). Examples of the synergistic interaction of cytokines are the stimulation of inflammatory reactions (IL-1, IL-6 and TNFa), as well as the synthesis of IgE

(IL-4, IL-5 and IL-13).

Some cytokines induce the synthesis of other cytokines (cascade). The cascading action of cytokines is necessary for the development of inflammatory and immune reactions. The ability of some cytokines to enhance or weaken the production of others determines important positive and negative regulatory mechanisms.

The antagonistic effect of cytokines is known, for example, the production of IL-6 in response to an increase in the concentration of TNFa can be

a negative regulatory mechanism for controlling the production of this mediator during inflammation.

Cytokine regulation of target cell functions is carried out using autocrine, paracrine or endocrine mechanisms. Some cytokines (IL-1, IL-6, TNFα, etc.) are capable of participating in the implementation of all of these mechanisms.

The cell's response to the influence of a cytokine depends on several factors:

On the type of cells and their initial functional activity;

From the local concentration of the cytokine;

From the presence of other mediator molecules.

Thus, producer cells, cytokines and their specific receptors on target cells form a single mediator network. It is the set of regulatory peptides, and not individual cytokines, that determine the final response of the cell. Currently, the cytokine system is considered as a universal regulatory system at the level of the whole organism, ensuring the development of protective reactions (for example, during infection).

In recent years, an idea has emerged of a cytokine system that combines:

1) producer cells;

2) soluble cytokines and their antagonists;

3) target cells and their receptors (Fig. 7.1).

Disturbances in various components of the cytokine system lead to the development of numerous pathological processes, and therefore identification of defects in this regulatory system is important for correct diagnosis and prescription of adequate therapy.

First, let's look at the main components of the cytokine system.

Cytokine producing cells

I. The main group of cytokine-producing cells in the adaptive immune response are lymphocytes. Resting cells do not secrete cytokines. Upon antigen recognition and with the participation of receptor interactions (CD28-CD80/86 for T lymphocytes and CD40-CD40L for B lymphocytes), cell activation occurs, leading to the transcription of cytokine genes, translation and secretion of glycosylated peptides into the intercellular space.

Rice. 7.1. Cytokine system

CD4 T helper cells are represented by subpopulations: Th0, Th1, Th2, Th17, Tfh, which differ from each other in the spectrum of secreted cytokines in response to various antigens.

Th0 produce a wide range of cytokines in very low concentrations.

Direction of differentiation Th0 determines the development of two forms of immune response with a predominance of humoral or cellular mechanisms.

The nature of the antigen, its concentration, localization in the cell, the type of antigen-presenting cells and a certain set of cytokines regulate the direction of Th0 differentiation.

Dendritic cells, after uptake and processing of antigen, present antigenic peptides to Th0 cells and produce cytokines that regulate the direction of their differentiation into effector cells. The role of individual cytokines in this process is shown in Fig. 7.2. IL-12 induces the synthesis of IFNγ by T lymphocytes and hGC. IFN ensures the differentiation of Th1, which begin to secrete cytokines (IL-2, IFN, IL-3, TNF-a, lymphotoxins) that regulate the development of reactions to intracellular pathogens

(delayed hypersensitivity (DTH) and various types of cellular cytotoxicity).

IL-4 ensures the differentiation of Th0 into Th2. Activated Th2 produce cytokines (IL-4, IL-5, IL-6, IL-13, etc.) that determine the proliferation of B lymphocytes, their further differentiation into plasma cells, and the development of antibody reactions, mainly to extracellular pathogens.

IFN negatively regulates the function of Th2 cells and, conversely, IL-4, IL-10, secreted by Th2, inhibit the function of Th1 (Fig. 7.3). The molecular mechanism of this regulation is associated with transcription factors. The expression of T-bet and STAT4, determined by IFNu, directs the differentiation of T cells along the Th1 pathway and suppresses the development of Th2. IL-4 induces the expression of GATA-3 and STAT6, which accordingly ensures the conversion of naive Th0 into Th2 cells (Fig. 7.2).

In recent years, a special subpopulation of helper T cells (Th17) producing IL-17 has been described. Members of the IL-17 family can be expressed by activated memory cells (CD4CD45RO), γ5T cells, NKT cells, neutrophils, monocytes under the influence of IL-23, IL-6, TGFβ produced by macrophages and dendritic cells. The main differentiation factor in humans is ROR-C, in mice it is ROR-γ l The cardinal role of IL-17 in the development of chronic inflammation and autoimmune pathology has been shown (see Fig. 7.2).

In addition, T cells in the thymus can differentiate into natural regulatory cells (Tregs) expressing CD4 + CD25 + surface markers and the transcription factor FOXP3. These cells are able to suppress the immune response mediated by Th1 and Th2 cells through direct cell-to-cell contact and the synthesis of TGFβ and IL-10.

Schemes of differentiation of Th0 clones and the cytokines they secrete are presented in Fig. 7.2 and 7.3 (see also color insert).

T-cytotoxic cells (CD8 +), natural killer cells, are weak producers of cytokines such as interferons, TNF-a and lymphotoxins.

Excessive activation of one of the Th subpopulations can determine the development of one of the variants of the immune response. Chronic imbalance of Th activation can lead to the formation of immunopathological conditions associated with the manifestations of

mi allergies, autoimmune pathology, chronic inflammatory processes, etc.

Rice. 7.2. Various subsets of T lymphocytes that produce cytokines

II. In the innate immune system, the main producers of cytokines are myeloid cells. Using Toll-like receptors (TLRs), they recognize similar molecular structures of various pathogens, the so-called pathogen-associated molecular patterns (PAMPs), for example, lipopolysaccharide (LPS) of Gram-negative bacteria, lipoteichoic acids, peptidoglycans of Gram-positive microorganisms, flagellin, DNA rich in unmethylated CpGs repetitions, etc. As a result

This interaction with TLR triggers an intracellular signal transduction cascade, leading to the expression of genes for two main groups of cytokines: pro-inflammatory and type 1 IFN (Fig. 7.4, see also color insert). Mainly these cytokines (IL-1, -6, -8, -12, TNFa, GM-CSF, IFN, chemokines, etc.) induce the development of inflammation and are involved in protecting the body from bacterial and viral infections.

Rice. 7.3. Spectrum of cytokines secreted by Th1 and Th2 cells

III. Cells not related to the immune system (connective tissue cells, epithelium, endothelium) constitutively secrete autocrine growth factors (FGF, EGF, TGFr, etc.). and cytokines that support the proliferation of hematopoietic cells.

Cytokines and their antagonists are described in detail in a number of monographs (Kovalchuk L.V. et al., 2000; Ketlinsky S.A., Simbirtsev A.S.,

Rice. 7.4. TLR-mediated induction of cytokine production by innate immune cells

Excessive expression of cytokines is unsafe for the body and can lead to the development of an excessive inflammatory reaction, an acute phase response. Various inhibitors are involved in the regulation of the production of proinflammatory cytokines. Thus, a number of substances have been described that nonspecifically bind the cytokine IL-1 and prevent the manifestation of its biological action (a2-macroglobulin, C3-component of complement, uromodulin). Specific inhibitors of IL-1 include soluble decoy receptors, antibodies, and IL-1 receptor antagonist (IL-1RA). With the development of inflammation, the expression of the IL-1RA gene increases. But even normally, this antagonist is present in the blood in high concentrations (up to 1 ng/ml or more), blocking the action of endogenous IL-1.

Target cells

The action of cytokines on target cells is mediated through specific receptors that bind cytokines with very high affinity, and individual cytokines can use

common receptor subunits. Each cytokine binds to its specific receptor.

Cytokine receptors are transmembrane proteins and are divided into 5 main types. The most common is the so-called hematopoietin type of receptors, which have two extracellular domains, one of which contains a common sequence of amino acid residues of two repeats of tryptophan and serine, separated by any amino acid (WSXWS motif). The second type of receptor may have two extracellular domains with a large number of conserved cysteines. These are receptors of the IL-10 and IFN family. The third type is represented by cytokine receptors belonging to the TNF group. The fourth type of cytokine receptors belongs to the superfamily of immunoglobulin receptors, which have extracellular domains that resemble the domains of immunoglobulin molecules in structure. The fifth type of receptors that bind molecules of the chemokine family is represented by transmembrane proteins that cross the cell membrane in 7 places. Cytokine receptors can exist in a soluble form, retaining the ability to bind ligands (Ketlinsky S.A. et al., 2008).

Cytokines can influence the proliferation, differentiation, functional activity and apoptosis of target cells (see Fig. 7.1). The manifestation of the biological activity of cytokines in target cells depends on the participation of various intracellular systems in signal transmission from the receptor, which is associated with the characteristics of the target cells. The signal for apoptosis is carried out, among other things, using a specific region of the TNF receptor family, the so-called “death” domain (Fig. 7.5, see color insert). Differentiation and activating signals are transmitted through intracellular Jak-STAT proteins - signal transducers and transcription activators (Fig. 7.6, see color insert). G proteins are involved in signal transduction from chemokines, which leads to increased cell migration and adhesion.

A comprehensive cytokine system analysis includes the following.

I. Evaluation of producer cells.

1. Determination of expression:

Receptors recognizing a pathogen or antigen TCR, TLR) at the level of genes and protein molecules (PCR, flow cytometry method);

Adapter molecules that conduct a signal that triggers the transcription of cytokine genes (PCR, etc.);

Rice. 7.5. Signal transmission from the TNF receptor

Rice. 7.6. Jak-STAT - cytokine receptor type 1 signaling pathway

Cytokine genes (PCR); protein molecules of cytokines (assessment of the cytokine-synthesizing function of human mononuclear cells).

2. Quantitative determination of cell subpopulations containing certain cytokines: Th1, Th2 Th17 (method of intracellular staining of cytokines); determination of the number of cells secreting certain cytokines (ELISPOT method, see Chapter 4).

II. Assessment of cytokines and their antagonists in biological environments of the body.

1. Testing the biological activity of cytokines.

2. Quantitative determination of cytokines using ELISA.

3. Immunohistochemical staining of cytokines in tissues.

4. Determination of the ratio of opposite cytokines (pro- and anti-inflammatory), cytokines and cytokine receptor antagonists.

III. Evaluation of target cells.

1. Determination of the expression of cytokine receptors at the level of genes and protein molecules (PCR, flow cytometry method).

2. Determination of signaling molecules in intracellular contents.

3. Determination of the functional activity of target cells.

Currently, numerous methods have been developed to assess the cytokine system, which provide diverse information. Among them are:

1) molecular biological methods;

2) methods for the quantitative determination of cytokines using immunoassays;

3) testing the biological activity of cytokines;

4) intracellular cytokine staining;

5) the ELISPOT method, which allows the detection of cytokines around a single cytokine-producing cell;

6) immunofluorescence.

We provide a brief description of these methods.

By using molecular biological methods You can study the expression of cytokine genes, their receptors, signaling molecules, and study the polymorphism of these genes. In recent years, a large number of studies have been carried out that have revealed associations between variant alleles of genes for molecules of the cytokine system and predisposition

to a number of diseases. The study of allelic variants of cytokine genes can provide information about the genetically programmed production of a particular cytokine. The most sensitive is considered to be the real-time polymerase chain reaction - RT-PCR (see Chapter 6). Hybridization method in situ allows you to clarify the tissue and cellular localization of cytokine gene expression.

Quantitative determination of cytokines in biological fluids and in cultures of peripheral blood mononuclear cells by ELISA can be characterized as follows. Since cytokines are local mediators, it is more appropriate to measure their levels in relevant tissues after extraction of tissue proteins or in natural fluids, such as tears, cavities, urine, amniotic fluid, cerebrospinal fluid, etc. Cytokine levels in serum or other body fluids reflect the current state of the immune system, i.e. synthesis of cytokines by body cells in vivo.

Determination of the levels of cytokine production by peripheral blood mononuclear cells (PBMCs) shows the functional state of the cells. Spontaneous production of cytokines by MNCs in culture indicates that the cells are already activated in vivo. The synthesis of cytokines induced (by various stimulants, mitogens) reflects the potential, reserve ability of cells to respond to an antigenic stimulus (in particular, to the action of drugs). Reduced induced production of cytokines may serve as one of the signs of an immunodeficiency state. Cytokines are not specific for a particular antigen. Therefore, specific diagnosis of infectious, autoimmune and allergic diseases by determining the level of certain cytokines is impossible. At the same time, assessing the levels of cytokines allows us to obtain data on the severity of the inflammatory process, its transition to the systemic level and prognosis, the functional activity of cells of the immune system, the ratio of Th1 and Th2 cells, which is very important in the differential diagnosis of a number of infectious and immunopathological processes.

In biological media, cytokines can be quantified using a variety of immunoassay methods, using polyclonal and monoclonal antibodies (see Chapter 4). ELISA allows you to find out what the exact concentrations of cytokines are in bio-

logical fluids of the body. Enzyme-linked immunosorbent detection of cytokines has a number of advantages over other methods (high sensitivity, specificity, independence from the presence of antagonists, the possibility of accurate automated recording, standardization of recording). However, this method also has its limitations: ELISA does not characterize the biological activity of cytokines and can give false results due to cross-reacting epitopes.

Biological testing carried out on the basis of knowledge of the basic properties of cytokines and their effects on target cells. The study of the biological effects of cytokines has led to the development of four types of cytokine testing:

1) by inducing proliferation of target cells;

2) by cytotoxic effect;

3) by inducing differentiation of bone marrow precursors;

4) for antiviral action.

IL-1 is determined by its stimulating effect on the proliferation of mouse thymocytes activated by mitogen in vitro; IL-2 - by its ability to stimulate the proliferative activity of lymphoblasts; TNF-α and lymphotoxins are tested for cytotoxic effects on mouse fibroblasts (L929). Colony-stimulating factors are assessed by their ability to support the growth of bone marrow precursors as colonies in agar. The antiviral activity of IFN is detected by the inhibition of the cytopathic effect of viruses in the culture of diploid human fibroblasts and the tumor line of mouse fibroblasts L-929.

Cell lines have been created whose growth depends on the presence of certain cytokines. In table Table 7.1 provides a list of cell lines used for cytokine testing. Based on the ability to induce proliferation of sensitive target cells, biotesting is carried out for IL-1, IL-2, IL-4, IL-6, IL-7, IL-15, etc. However, these testing methods are characterized by insufficient sensitivity and information content. Inhibitor and antagonist molecules can mask the biological activity of cytokines. Some cytokines exhibit general biological activity. However, these methods are ideal for testing the specific activity of recombinant cytokines.

Table 7.1. Cell lines used to test cytokine biological activity

End of table. 7.1

Lab 7-1

Determination of the biological activity of IL-1 by its comitogenic effect on the proliferation of mouse thymocytes

The method of biological testing of IL-1 is based on the ability of the cytokine to stimulate the proliferation of mouse thymocytes.

IL-1 can be determined in a culture of monocytes stimulated with LPS, as well as in any biological fluid of the body. It is necessary to pay attention to a number of details.

1. For testing, thymocytes of mice of the C3H/HeJ line are used, stimulated to proliferation with mitogens (concanavalin A - ConA and phytohemagglutinin - PHA). C3H/HeJ thymocytes were not chosen at random: mice of this inbred strain do not respond to LPS, which may be present in the test material and cause the production of IL-1.

2. Thymocytes respond to IL-2 and mitogens, therefore, the presence of IL-2 and mitogens should also be determined in preparations tested for IL-1.

Operating procedure

1. A suspension of thymocytes is obtained at a concentration of 12×10 6 /ml of RPMI 1640 medium containing 10% fetal bovine serum and 2-mercaptoethanol (5×10 -5 M).

2. Prepare a series of serial two-fold dilutions of experimental (biological body fluids) and control samples. Biological fluids containing IL-1 or samples obtained by incubation of mononuclear cells without LPS, and a laboratory standard IL-1-containing preparation are used as controls. In 96-well round-bottomed plates, 50 µl of each dilution is transferred into 6 wells.

3. Add 50 μl of purified PHA (Wellcome) dissolved in complete medium at a concentration of 3 μg/ml to three wells of each dilution, and 50 μl of medium to the other 3 wells.

4. Add 50 μl of thymocyte suspension to each well and incubate for 48 hours at 37 °C.

6. Before completing the cultivation, 50 μl of a solution (1 μCi/ml) of [" 3 H]-thymidine is added to the wells and incubated for another 20 hours.

7. To determine the level of radioactivity, culture cells are transferred to filter paper using an automatic cell collector, the filters are dried and the inclusion of the label is determined with a liquid scintillation counter.

8. The results are expressed as a stimulation factor.

where m cp is the average number of pulses in 3 wells.

If thymocytes respond to stimulation with standard IL-1, then the stimulation index of the test sample exceeding 3 reliably indicates IL-1 activity.

Bioassay is the only method to assess the functioning of the cytokine, but this method must be complemented by various types of appropriate monitoring for specificity using monoclonal antibodies. The addition of certain monoclonal antibodies to the cytokine into the culture blocks the biological activity of the cytokine, which proves that the signal for the proliferation of the cell line is the detectable cytokine.

Use of bioassays to detect interferon. The principle of assessing the biological activity of IFN is based on its antiviral effect, which is determined by the degree of inhibition of the proliferation of the test virus in cell culture.

Cells that are sensitive to the action of IFN can be used in the work: primary trypsinized chicken and human embryonic fibroblast cells, continuous cells of human diploid fibroblasts and a mouse cell culture (L929).

When assessing the antiviral effect of IFN, it is advisable to use viruses with a short reproduction cycle and high sensitivity to the action of IFN: mouse encephalomyelitis virus, mouse vesicular stomatitis virus, etc.

Lab 7-2

Determination of interferon activity

1. A suspension of diploid human fetal fibroblasts on a medium with 10% bovine fetal serum (cell concentration - 15-20×10 6 /ml) is poured into sterile 96-well flat-bottomed plates, 100 µl per well and placed in a CO 2 incubator at a temperature 37 °C.

2. After the formation of a complete monolayer, the growth medium is removed from the wells and 100 μl of maintenance medium is added to each well.

3. Titration of IFN activity in the studied samples is carried out using the method of two-fold dilutions on a monolayer of fibroblasts.

Simultaneously with the samples, mouse encephalomyelitis virus (MEV) is introduced into the wells at a dose that causes 100% cell damage 48 hours after infection.

4. For control, use wells with intact (untreated) cells infected with the virus.

In each study, reference IFN samples with known activity are used as reference drugs.

5. Plates with sample dilutions are incubated for 24 hours at 37 °C in an atmosphere with 5% CO 2 content.

6. The level of IFN activity is determined by the reciprocal of the maximum dilution of the test sample, which delays the cytopathic effect of the virus by 50%, and is expressed in units of activity per 1 ml.

7. To determine the type of IFN, antiserum against IFNα, IFNβ or IFNγ is added to the system. The antiserum cancels the action of the corresponding cytokine, which makes it possible to identify the type of IFN.

Determination of biological activity of inhibitory factor migration. Currently, completely new ideas have been formed about the nature and properties of MIF, discovered in the 60s of the last century as a mediator of cellular immunity and which remained without due attention for many years (Bloom B.R., Bennet B., 1966; David J.R., 1966). Only in the last 10-15 years has it become clear: MIF is one of the most important biological mediators in the body with a wide range of biological functions as a cytokine, hormone, and enzyme. The effect of MIF on target cells is realized through the CD74 - receptor or through the non-classical endocytosis pathway.

MIF is considered an important mediator of inflammation, activating the function of macrophages (cytokine production, phagocytosis, cytotoxicity, etc.), as well as an endogenous immunoregulatory hormone that modulates glucocorticoid activity.

More and more information is accumulating about the role of MIF in the pathogenesis of many inflammatory diseases, including sepsis, rheumatoid arthritis (RA), glomerulonephritis, etc. In RA, the concentration of MIF in the fluid of the affected joints is significantly increased, which correlates with the severity of the disease. Under the influence of MIF, the production of pro-inflammatory cytokines by both macrophages and synovial cells increases.

Various methods are known for testing the activity of MIF, where migrating cells (target cells for MIF) are placed in a glass capillary (capillary test), in a drop of agarose, or in an agarose well.

We present a relatively simple screening method based on the formation of cell microcultures (leukocytes or macrophages), standard in area and number of cells, at the bottom of the wells of a 96-well flat-bottomed plate, followed by their cultivation in a nutrient medium and determination of changes in the area of these microcultures under the influence of MIF ( Suslov A.P., 1989).

Lab 7-3

Definition of MIF activity

Determination of the biological activity of MIF is carried out using a device for the formation of cell microcultures (Fig. 7.7) - MIGROSKRIN (N.F. Gamaleya Research Institute of Epidemiology and Microbiology of the Russian Academy of Medical Sciences).

1. Add 100 µl of a sample diluted in a culture medium, in which MIF activity is determined (each dilution in 4 parallels, experimental samples), into the wells of a 96-well plate (Flow, UK or similar). The culture medium includes RPMI 1640, 2 mM L-glutamine, 5% fetal bovine serum, 40 μg/ml gentamicin.

2. Add culture medium (in 4 parallels) of 100 μl to control wells.

3. A cell suspension of peritoneal macrophages is prepared, for which 2 hybrid mice (CBAxC57B1/6)F1 are injected intraperitoneally with 10 ml of Hanks solution with heparin (10 U/ml), and the abdomen is gently massaged for 2-3 minutes. Then the animal is killed by decapitation, the abdominal wall is carefully pierced in the groin area, and the exudate is sucked out through a needle with a syringe. The cells of the peritoneal exudate are washed twice with Hanks' solution, centrifuging them for 10-15 minutes at 200 g. Then a cell suspension is prepared with a concentration of 10±1 million/ml of RPMI 1640 medium. Counting is carried out in a Goryaev chamber.

4. Assemble the MIGROSKRIN system, which is a stand for directional and standard fixation of tips with cell cultures in a strictly vertical position at a given height above the center of the well of a 96-well culture plate, and also includes 92 tips for automatic pipettes from Costar, USA (Fig. .7.7).

Insert the legs of the tripod into the corner wells of the tablet. The cell suspension is drawn into the tips with an automatic pipette - 5 μl each, rinsed to remove excess cells by dropping them once into the medium and inserted vertically into the sockets of the system stand. The filled rack with tips is kept at room temperature for 1 hour on a strictly horizontal surface. During this time, the suspension cells settle to the bottom of the wells, where standard cell microcultures are formed.

5. The stand with tips is carefully removed from the tablet. The cell microculture plate is placed in a strictly horizontal position in a CO 2 incubator, where it is cultured for 20 hours. During cultivation, the cells migrate along the bottom of the well.

6. Quantitative recording of the results after incubation is carried out using a binocular magnifying glass, visually assessing the size of the colony on a scale inside the eyepiece. Microcultures have the shape of a circle. The researchers then determine the average colony diameter by measuring colonies in 4 test or control wells. The measurement error is ±1 mm.

The migration index (MI) is calculated using the formula:

A sample has MIF activity if the IM values are equal

The conventional unit (AU) of MIF activity is taken to be the inverse value equal to the value of the highest dilution of the sample (sample), at which the migration index is 0.6 ± 0.2.

Biological activity of PEOα is assessed by its cytotoxic effect on the line of transformed fibroblasts L-929. Recombinant TNF-α is used as a positive control, and cells in the culture medium are used as a negative control.

Calculate the cytotoxic index (CI):

Where a- number of living cells in the control; b- the number of living cells in the experiment.

Rice. 7.7. Scheme MIGROSKRIN - devices for quantitative assessment of migration of cell cultures

The cells are stained with a dye (methylene blue), which is included only in dead cells.

The standard unit of TNF activity is taken to be the reciprocal dilution of the sample required to obtain 50% cellular cytotoxicity. Specific activity of a sample is the ratio of activity in arbitrary units per 1 ml to the concentration of protein contained in the sample.

Intracellular cytokine staining. Changes in the ratio of cells producing various cytokines may reflect the pathogenesis of the disease and serve as a criterion for prognosis of the disease and evaluation of therapy.

The intracellular staining method is used to determine cytokine expression at the single cell level. Flow cytometry allows you to count the number of cells expressing a particular cytokine.

Let us list the main stages of determining intracellular cytokines.

Unstimulated cells produce small amounts of cytokines, which, as a rule, are not stored, so an important step in the assessment of intracellular cytokines is stimulation of lymphocytes and blocking the release of these products from cells.

The most commonly used cytokine inducer is the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) in combination with the calcium ionophore ionomycin (IN). The use of this combination causes the synthesis of a wide range of cytokines: IFN, IL-4, IL-2, TNFα. The disadvantage of using FMA-IN is the problem of identifying CD4 molecules on the surface of lymphocytes after such activation. Also, the production of cytokines by T lymphocytes is induced using mitogens (PHA). B cells and monocytes stimulate

Mononuclear cells are incubated in the presence of inducers of cytokine production and a blocker of their intracellular transport, brefeldin A or monensin, for 2-6 hours.

The cells are then resuspended in a buffer solution. For fixation, add 2% formaldehyde and incubate for 10-15 minutes at room temperature.

Then the cells are treated with saponin, which increases the permeability of the cell membrane, and stained with monoclonal antibodies specific to the detected cytokines. Preliminary staining of surface markers (CD4, CD8) increases the amount of information obtained about the cell and makes it possible to more accurately determine its population affiliation.

There are some limitations in the application of the methods described above. Thus, with their help it is impossible to analyze the synthesis of cytokines by a single cell, it is impossible to determine the number of cytokine-producing cells in a subpopulation, it is impossible to determine whether cytokine-producing cells express unique markers, whether different cytokines are synthesized by different cells or by the same ones. The answer to these questions is obtained using other research methods. To determine the frequency of cytokine-producing cells in a population, the limiting dilution method and a variant of the ELISPOT enzyme-linked immunosorbent assay are used (see Chapter 4).

In situ hybridization method. The method includes:

2) fixation with paraformaldehyde;

3) detection of mRNA using labeled cDNA. In some cases, cytokine mRNA is determined on sections using radioisotope PCR.

Immunofluorescence. The method includes:

1) freezing the organ and preparing cryostat sections;

2) fixation;

3) treatment of sections with fluorescein-labeled anti-cytokine antibodies;

4) visual observation of fluorescence.

These techniques (hybridization in situ and immunofluorescence) are fast and do not depend on threshold concentrations of the secreted product. However, they do not measure the amount of cytokine secreted and can be technically challenging. Various close monitoring for nonspecific reactions is necessary.

Using the presented methods for assessing cytokines, pathological processes associated with disturbances in the cytokine system at various levels were identified.

Thus, assessment of the cytokine system is extremely important for characterizing the state of the body's immune system. The study of various levels of the cytokine system allows us to obtain information about the functional activity of different types of immunocompetent cells, the severity of the inflammatory process, its transition to the systemic level and the prognosis of the disease.

Questions and tasks

1. List the general properties of cytokines.

2. Give the classification of cytokines.

3. List the main components of the cytokine system.

4. List the cells that produce cytokines.

5. Describe the cytokine receptor families.

6. What are the mechanisms of functioning of the cytokine network?

7. Explain the production of cytokines in the innate immune system.

8. What are the main approaches to a comprehensive assessment of the cytokine system?

9. What are the methods for testing cytokines in body fluids?

10. What are the defects in the cytokine system in various pathologies?

11. What are the main methods for biological testing of IL-1, IFN, MIF, TNFa in biological fluids?

12. Describe the process of determining the intracellular content of cytokines.

13. Describe the process of determining the cytokines secreted by a single cell.

14. Describe the sequence of methods used to identify a defect at the level of the cytokine receptor.

15. Describe the sequence of methods used to identify a defect at the level of cytokine-producing cells.

16. What information can be obtained by studying the production of cytokines in a culture of mononuclear cells in blood serum?

Activation of cells in the inflammatory zone is manifested in the fact that the cells begin to synthesize and secrete many cytokines that affect nearby cells and cells of distant organs. Among all these cytokines, there are those that promote (pro-inflammatory) and those that prevent the development of the inflammatory process (anti-inflammatory). Cytokines cause effects similar to manifestations of acute and chronic infectious diseases.

Pro-inflammatory cytokines

90% of lymphocytes (a type of white blood cell) and 60% of tissue macrophages (cells capable of capturing and digesting bacteria) are capable of secreting pro-inflammatory cytokines. Stimulators of cytokine production are pathogens and cytokines themselves (or other inflammatory factors).

Local release of proinflammatory cytokines causes the formation of a focus of inflammation. With the help of specific receptors, pro-inflammatory cytokines bind and involve other types of cells in the process: skin, connective tissue, inner walls of blood vessels, epithelial cells. All these cells also begin to produce pro-inflammatory cytokines.

The most important proinflammatory cytokines are IL-1 (interleukin-1) and TNF-alpha (tumor necrosis factor-alpha). They cause the formation of foci of adhesion (sticking) on the inner lining of the vascular wall: first, leukocytes adhere to the endothelium and then penetrate through the vascular wall.

These pro-inflammatory cytokines stimulate the synthesis and release of other pro-inflammatory cytokines (IL-8 and others) by leukocytes and endothelial cells and thereby activate the cells to produce inflammatory mediators (leukotrienes, histamine, prostaglandins, nitric oxide and others).

When an infection enters the body, the production and release of IL-1, IL-8, IL-6, TNF-alpha begins at the site of introduction of the microorganism (in the cells of the mucous membrane, skin, regional lymph nodes) - that is, cytokines activate local protective reactions.

Both TNF-alpha and IL-1, in addition to local effects, also have a systemic effect: they activate the immune system, endocrine, nervous and hematopoietic systems. Proinflammatory cytokines can cause about 50 different biological effects. Almost all tissues and organs can be their targets.

For example, anemia in acute and chronic infectious diseases is the result of exposure to proinflammatory cytokines (interleukin-1, interferon-beta, interferon-gamma, TNF, neopterin) on the body. They suppress the proliferation of erythroid lineage, the release of iron from macrophage cells and inhibit the production of erythropoietin in the kidneys. Cytokines act very effectively and quickly.

Anti-inflammatory cytokines

The action of pro-inflammatory cytokines is controlled by anti-inflammatory cytokines, which include IL-4, IL-13, IL-10, TGF-beta. They can not only suppress the synthesis of proinflammatory cytokines, but also promote the synthesis of receptor antagonists of interleukins (RAIL).

The relationship between anti-inflammatory and pro-inflammatory cytokines is an important point in regulating the occurrence and development of the inflammatory process. The course of the disease and its outcome depend on this balance. It is cytokines that stimulate the production of blood clotting factors in vascular endothelial cells, the production of chondrolytic enzymes, and promote the formation of scar tissue.

Cytokines and the immune response

All cells in the immune system have certain distinct functions. Their coordinated interaction is carried out by cytokines - regulators of immune reactions. They ensure the exchange of information between cells of the immune system and coordination of their actions.

The set and quantity of cytokines is a matrix of signals (often changing) that act on cell receptors. The complex nature of these signals is explained by the fact that each cytokine can suppress or activate several processes (including the synthesis of its own or other cytokines) and the formation of receptors on the cell surface.

Cytokines provide a relationship within the immune system between specific immunity and the body’s nonspecific defense reaction, between humoral and cellular immunity. It is cytokines that communicate between phagocytes (providing cellular immunity) and lymphocytes (cells of humoral immunity), as well as between lymphocytes of different functions.

Through cytokines, T-helpers (lymphocytes that “recognize” foreign proteins of microorganisms) transmit a command to T-killers (cells that destroy foreign proteins). In the same way, with the help of cytokines, suppressor T cells (a type of lymphocyte) control the function of killer T cells and transmit information to them to stop cell destruction.

If such a connection is broken, then the death of cells (already native to the body, and not foreign) will continue. This is how autoimmune diseases develop: the synthesis of IL-12 is not controlled, the cell-mediated immune response will be overactive.

The course and outcome of an infectious disease depends on the ability of its pathogen (or its components) to induce the synthesis of the cytokine IL-12. For example, the fungal species Candida albicans can induce the synthesis of IL-12, which contributes to the development of effective cellular defense against this pathogen. Leishmania suppresses the synthesis of IL-12 - a chronic infection develops. HIV suppresses the synthesis of IL-12, and this leads to defects in cellular immunity in AIDS.

Cytokines also regulate the body’s specific immune response to the introduction of a pathogen. If local protective reactions have failed, then cytokines act at the systemic level, that is, they affect all systems and organs that are involved in maintaining homeostasis.

When they influence the central nervous system, the entire complex of behavioral reactions changes, the synthesis of most hormones, protein synthesis and plasma composition changes. But all the changes that occur are not random: they are either necessary to increase protective reactions, or contribute to the switching of the body’s energy to fight pathogenic effects.

It is cytokines, communicating between the endocrine, nervous, hematopoietic and immune systems, that involve all these systems in the formation of a complex protective reaction of the body to the introduction of a pathogenic agent.

Macrophage engulfs bacteria and releases cytokines (3D model) - video

Cytokine gene polymorphism analysis

Cytokine gene polymorphism analysis is a genetic study at the molecular level. Such studies provide a wide range of information that makes it possible to identify the presence of polymorphic genes (pro-inflammatory variants) in the person being examined, predict predisposition to various diseases, develop a program for the prevention of such diseases for this particular person, etc.

In contrast to single (sporadic) mutations, polymorphic genes are found in approximately 10% of the population. Carriers of such polymorphic genes have increased activity of the immune system during surgical interventions, infectious diseases, and mechanical effects on tissue. The immunogram of such individuals often reveals a high concentration of cytotoxic cells (killer cells). Such patients more often experience septic, purulent complications of diseases.

But in some situations, such increased activity of the immune system can interfere: for example, during in vitro fertilization and embryo transfer. And the combination of pro-inflammatory genes interleukin-1 or IL-1 (IL-1), receptor antagonist of interleukin-1 (RAIL-1), tumor necrosis factor-alpha (TNF-alpha) is a predisposing factor for miscarriage during pregnancy. If the examination reveals the presence of pro-inflammatory cytokine genes, then special preparation for pregnancy or IVF (in vitro fertilization) is required.

Cytokine profile analysis includes detection of 4 polymorphic gene variants:

interleukin 1-beta (IL-beta);
interleukin-1 receptor antagonist (ILRA-1);
interleukin-4 (IL-4);
tumor-necrotic factor-alpha (TNF-alpha).

No special preparation is required to take the test. The material for the study is a scraping from the buccal mucosa.

Modern studies have shown that with recurrent miscarriage, genetic factors of thrombophilia (a tendency to form blood clots) are often found in women's bodies. These genes can lead not only to miscarriage, but also to placental insufficiency, fetal growth retardation, and late toxicosis.

In some cases, the polymorphism of thrombophilia genes in the fetus is more pronounced than in the mother, since the fetus also receives genes from the father. Mutations of the prothrombin gene lead to almost one hundred percent intrauterine fetal death. Therefore, especially complex cases of miscarriage require examination and the husband.

An immunological examination of the husband will help not only determine the prognosis of pregnancy, but will also identify risk factors for his health and the possibility of using preventive measures. If risk factors are identified in the mother, it is advisable to then conduct an examination of the child - this will help develop an individual program for the prevention of diseases in the child.

In case of infertility, it is advisable to identify all currently known factors that can lead to it. A complete genetic study of gene polymorphism includes 11 indicators. The examination can help identify a predisposition to placental dysfunction, high blood pressure, and preeclampsia. Accurate diagnosis of the causes of infertility will allow the necessary treatment to be carried out and will make it possible to maintain the pregnancy.

An extended hemostasiogram can provide information not only for obstetric practice. Using the study of gene polymorphism, it is possible to identify genetic factors of predisposition to the development of atherosclerosis and coronary heart disease, predict its course and the likelihood of developing myocardial infarction. Even the probability of sudden death can be calculated using genetic research.

The influence of gene polymorphism on the rate of development of fibrosis in patients with chronic hepatitis C has also been studied, which can be used in predicting the course and outcome of chronic hepatitis.

Molecular genetic studies of multifactorial diseases help not only in creating an individual health prognosis and preventive measures, but also in the development of new treatment methods using anti-cytokine and cytokine drugs.

Cytokine therapy

Treatment of tumor diseases

Cytokine therapy can be used at any (even IV) stage of a malignant disease, in the presence of severe concomitant pathology (hepatic-renal or cardiovascular failure). Cytokines selectively destroy only malignant tumor cells and do not affect healthy ones. Cytokine therapy can be used as an independent treatment method or as part of complex therapy.

Immunological studies in cancer patients have shown that most malignant diseases are accompanied by impaired immunological response. The degree of suppression depends on the size of the tumor and the treatment performed (radiation therapy and chemotherapy). Data were obtained on the biological effects of cytokines (interleukin-2, interferons, tumor-necrotic factor and others).

Cytokine therapy has been used in oncology for several decades. But previously, interleukin-2 (IL-2) and interferon-alpha (IFN-alpha) were used mainly - effective only for skin melanoma and kidney cancer. In recent years, new drugs have been created, and the indications for their effective use have expanded.

One of the cytokine drugs, tumor necrosis factor (TNF-alpha), acts through receptors located on the malignant cell. This cytokine is produced in the human body by monocytes and macrophages. When interacting with the receptors of a malignant cell, the cytokine triggers the program of death of this cell.

TNF-alpha began to be used in oncological practice in the USA and Europe back in the 80s. It is still in use today. But the high toxicity of the drug limits its use only to those cases where it is possible to isolate the organ with the tumor process from the general bloodstream (kidneys, limbs). In this case, the drug circulates using a heart-lung machine only in the affected organ, and does not enter the general bloodstream.

In Russia, in 1990, the drug Refnot (TNF-T) was created due to the fusion of the Thymosin-alpha and Tumor Necrosis Factor genes. It is 100 times less toxic than TNF, has undergone clinical trials and since 2009 has been approved for use in the treatment of various types and locations of malignant tumors.

Given the reduced toxicity of the drug, it can be administered intramuscularly or subcutaneously. The drug has an effect on both the primary tumor site and metastases (including distant ones), in contrast to the TNF-alpha drug, which could only have an effect on the primary site.

Another promising cytokine drug is Interferon-gamma (IFN-gamma). On its basis, the drug Ingaron was created in Russia in 1990. It has a direct effect on tumor cells or triggers an apoptosis program (the cell itself programs and carries out its death), and increases the efficiency of immune cells.

The drug has also undergone clinical trials and since 2005 has been approved for use in the treatment of malignant tumors. The drug activates those receptors on the malignant cell, with which Refnot then interacts. Therefore, cytokine therapy with Refnot is most often combined with the use of Ingaron.

The method of administration of these drugs (intramuscular or subcutaneous) allows treatment to be carried out on an outpatient basis. Cytokine therapy is contraindicated only during pregnancy and autoimmune diseases. In addition to the direct effect on the malignant cell, Ingaron and Refnot have an indirect effect - they activate their own cells of the immune system (T-lymphocytes and phagocytes), and increase overall immunity.

Unfortunately, the effectiveness of cytokine therapy is only 30-60%, depending on the stage and location of the tumor, the type of malignancy, the extent of the process, and the general condition of the patient. The higher the stage of the disease, the less pronounced the effect of treatment.

But even in the presence of multiple and distant metastases and the impossibility of chemotherapy (due to the severity of the patient’s general condition), positive results are noted in the form of an improvement in general well-being and a halt in the further development of the disease.

The main directions of action of modern cytokine drugs:

direct effect on the cells of the tumor itself and metastases;
enhancing the antitumor effect of chemotherapy;
prevention of metastases and tumor relapses;
reduction of adverse reactions of chemotherapy by suppression of hematopoiesis and immunosuppression;
treatment and prevention of infectious complications during treatment.

Possible results of using cytokine therapy:

complete disappearance of the tumor or reduction in its size (due to the initiation of apoptosis - programmed death of tumor cells);
stabilization of the process or partial regression of the tumor (when cell cycle arrest is triggered in tumor cells);
no effect – tumor growth and metastasis continues (if tumor cells are insensitive to the drug due to mutations).

From the above it is clear that the clinical result of the use of cytokine therapy depends on the characteristics of the tumor cells in the patient himself. To assess the effectiveness of the use of cytokines, 1-2 courses of treatment are carried out and the dynamics of the process are assessed using various instrumental examination methods.

The possibility of using cytokine therapy does not mean abandoning other treatment methods (surgery, chemotherapy or radiation therapy). Each of them has its own advantages on the tumor. All indicated and available treatment methods should be used in each specific case.

Cytokines significantly facilitate the tolerability of radiation and chemotherapy, prevent the occurrence of neutropenia (decrease in the number of leukocytes) and the development of infections during chemoradiotherapy. In addition, Refnot increases the effectiveness of most chemotherapy drugs. Using it in combination with Ingaron a week before starting chemotherapy and continuing to use the cytokine after chemotherapy will protect against infections or cure them without antibiotics.

The cytokine therapy regimen is prescribed to each patient individually. Both drugs exhibit virtually no toxicity (unlike chemotherapy drugs), have no side effects and are well tolerated by patients, do not have an inhibitory effect on hematopoiesis, and increase antitumor specific immunity.

Treatment of schizophrenia

Research has established that cytokines are involved in psychoneuroimmune reactions and ensure the combined functioning of the nervous and immune systems. The balance of cytokines regulates the process of regeneration of defective or damaged neurons. This is the basis for the use of new methods of treating schizophrenia - cytokine therapy: the use of immunotropic cytokine-containing drugs.

One way is to use anti-TNF-alpha and anti-IFN-gamma antibodies (anti-tumor necrosis factor-alpha and anti-interferon-gamma antibodies). The drug is administered intramuscularly for 5 days, 2 times a day. per day.

There is also a technique for using a composite solution of cytokines. It is administered in the form of inhalations using a nebulizer, 10 ml per 1 injection. Depending on the patient’s condition, the drug is administered every 8 hours in the first 3-5 days, then for 5-10 days - 1-2 r./day and then reducing the dose to 1 r. in 3 days for a long time (up to 3 months) with complete abolition of psychotropic drugs.

Intranasal use of a cytokine solution (containing IL-2, IL-3, GM-CSF, IL-1beta, IFN-gamma, TNF-alpha, erythropoietin) helps to increase the effectiveness of treatment in patients with schizophrenia (including during the first attack of the disease), more long and stable remission. These methods are used in clinics in Israel and Russia.

More about schizophrenia

Cytokines, by their nature, are proteins produced by cells of the immune system (often called “factors” in the literature). They participate in the differentiation of newborn cells of the immune system, endowing them with certain characteristics that are the source of diversity of immune cells, and also ensure intercellular interaction. To make this process easier to understand, the immune cell production process can be compared to a factory. At the first stage, identical cell blanks leave the conveyor, then at the second stage, with the help of various groups of cytokines, each cell is endowed with special functions and sorted into groups for subsequent participation in immune processes. This is how T-lymphocytes, B-lymphocytes, neutrophils, basophils, eosinophils, and monocytes are obtained from identical cells.

Of interest to science is the peculiarity of the effect of a cytokine on a cell, which gives rise to the production of other cytokines by that cell. That is, one cytokine triggers the reaction of producing others cytokines.

Cytokines, depending on their effect on immune cells, are divided into six groups:

Interferons
Interleukins
Colony-stimulating factors
Growth factors
Chemokines
Tumor necrosis factors

Interferons are cytokines produced by cells in response to viral infection or other stimuli. These proteins (cytokines) block virus reproduction in other cells and take part in immune cell-cell interaction.

The first type (has antiviral and antitumor effects):

interferon-alpha

interferon beta

Interferon-gamma

Interferons alpha and beta have a similar mechanism of action, but are produced by different cells.

Interferon-alpha is produced by mononuclear phagocytes. From this follows its name - “ leukocyte interferon».

Interferon-beta is produced by fibroblasts. Hence its name - “ fibroblast interferon».

Interferons of the first type have their own tasks:

Increase the production of interleukins (IL1)
Reduce the pH level in the intercellular environment with increasing temperature
Binds to healthy cells and protects them from viruses
Capable of inhibiting cell proliferation (growth) by blocking the synthesis of amino acids
In conjunction with natural killer cells, they induce or suppress (depending on the situation) the formation of antigens

Interferon-gamma is produced by T lymphocytes and natural killer cells. It bears the name “ immune interferon»

Interferon of the second type also has tasks:

Activates T-lymphocytes, B-lymphocytes, macrophages, neutrophils,
Inhibits the proliferation of thymocytes,
Strengthens cellular immunity and autoimmunity,
Regulates apoptosis of normal and infected cells.

Interleukins(abbreviated IL) are cytokines that regulate the interaction between leukocytes. Science has identified 27 interleukins.

Colony-stimulating factors are cytokines that regulate the division and differentiation of bone marrow stem cells and blood cell precursors. These cytokines are responsible for the ability of lymphocytes to form clones, and are also able to stimulate the functionality of cells outside the bone marrow.

Growth factors – regulate the growth, differentiation and functionality of cells in various tissues

The following growth factors have been discovered to date:

transforming growth factors alpha and beta
epidermal growth factor
fibroblast growth factor
platelet-derived growth factor
nerve cell growth factor
insulin-like growth factor
heparin binding growth factor
endothelial cell growth factor

The functions of transforming growth factor beta are considered the most studied. It is responsible for suppressing the growth and activity of T-lymphocytes, suppresses some functions of macrophages, neutrophils, and B-lymphocytes. Although this factor is classified as a growth factor, it is actually involved in the reverse process, that is, it suppresses the immune response (suppresses the functions of cells involved in immune defense) when the infection is eliminated and the work of immune cells is no longer necessary. It is under the influence of this factor that collagen synthesis and the production of immunoglobulin IgA during wound healing are enhanced, and memory cells are generated.

Chemokines are cytokines with low molecular weight. Their main function is to attract leukocytes from the bloodstream to the site of inflammation, as well as to regulate the mobility of leukocytes.

Tumor necrosis factors(abbreviated as TNF) are two types of cytokines (TNF-alpha and TNF-beta). The results of their action: the development of cachexia (an extreme degree of exhaustion of the body as a result of slowing down the activity of the enzyme, which promotes the accumulation of fat in the body); development of toxic shock; inhibition of apoptosis (cell death) of cells of the immune system, induction of apoptosis of tumor and other cells; platelet activation and wound healing; inhibition of angiogenesis (vascular proliferation) and fibrogenesis (degeneration of tissue into connective tissue), granulomatosis (formation of granulomas - proliferation and transformation of phagocytes) and many other results.

Cytokines - classification, role in the body, treatment (cytokine therapy), reviews, price

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The site provides reference information for informational purposes only. Diagnosis and treatment of diseases must be carried out under the supervision of a specialist. All drugs have contraindications. Consultation with a specialist is required!

What are cytokines?

Cytokines- These are specific hormone-like proteins that are synthesized by various cells in the body: cells of the immune system, blood cells, spleen, thymus, connective tissue and other types of cells. The bulk of cytokines are produced by lymphocytes.

Cytokines are low molecular weight soluble proteins that provide signal transmission between cells. The synthesized cytokine is released onto the cell surface and interacts with receptors of neighboring cells. In this way, the signal is transmitted from cell to cell.

The formation and release of cytokines lasts a short time and is clearly regulated. The same cytokine can be produced by different cells and have an effect on different cells (targets). Cytokines can enhance the effect of other cytokines, but they can also neutralize or weaken it.

Cytokines are active in very small concentrations. They play an important role in the development of physiological and pathological processes. Currently, cytokines are used in the diagnosis of many diseases and are used as therapeutic agents for tumor, autoimmune, infectious and psychiatric diseases.

Functions of cytokines in the body

The functions of cytokines in the body are multifaceted. In general, their activity can be characterized as ensuring interaction between cells and systems:

regulation of the duration and intensity of immune reactions (antitumor and antiviral defense of the body);
regulation of inflammatory reactions;
participation in the development of autoimmune reactions;
determination of cell survival;
participation in the mechanism of allergic reactions;
stimulation or inhibition of cell growth;
participation in the process of hematopoiesis;
ensuring functional activity or toxic effects on the cell;
consistency of reactions of the endocrine, immune and nervous systems;
maintaining homeostasis (dynamic constancy) of the body.

It has now been found that cytokines are regulators of not only the body’s immune response. At a minimum, their significance has the following basic components:

regulation of the fertilization process, organ formation (including the immune system) and their development;
regulation of normally occurring (physiological) functions of the body;
regulation of cellular and humoral immunity (local and systemic protective reactions);
regulation of processes of restoration (regeneration) of damaged tissues.

Classification of cytokines

Currently, more than 200 cytokines are already known, and more and more of them are being discovered every year. There are several classifications of cytokines.

Classification of cytokines according to the mechanism of biological action:
1. Cytokines that regulate inflammatory responses:

pro-inflammatory (interleukins 1, 2, 6, 8, interferon and others);
anti-inflammatory (interleukins 4, 10, and others).

2. Cytokines that regulate cellular immunity: interleukin-1 (IL-1 or IL-1), IL-12 (IL-12), IFN-gamma (IFN-gamma), TRF-beta and others).
3. Cytokines that regulate humoral immunity (IL-4, IL-5, IFN-gamma, TRF-beta and others).

Another classification divides cytokines into groups by the nature of the action:

Interleukins (IL-1 - IL-18) are regulators of the immune system (they ensure interaction within the system itself and its connections with other systems).
Interferons (IFN-alpha, beta, gamma) are antiviral immunoregulators.
Tumor necrosis factors (TNF-alpha, TNF-beta) – have a regulatory and toxic effect on cells.
Chemokines (MCP-1, RANTES, MIP-2, PF-4) – ensure the active movement of various types of leukocytes and other cells.
Growth factors (EGF, FGF, TGF-beta) – provide and regulate the growth, differentiation and functional activity of cells.
Colony-stimulating factors (G-CSF, M-CSF, GM-CSF) – stimulate the differentiation, growth and reproduction of hematopoietic sprouts (hematopoietic cells).

Interleukins from numbers 1 to 29 cannot be combined into one group based on their common function, since they include pro-inflammatory cytokines, differentiating cytokines for lymphocytes, growth and some regulatory ones.

Cytokines and inflammation

Pro-inflammatory cytokines

Macrophage engulfs bacteria and releases cytokines (3D model) - video

Cytokine gene polymorphism analysis

Cytokine profile analysis includes detection of 4 polymorphic gene variants:

interleukin 1-beta (IL-beta);
interleukin-1 receptor antagonist (ILRA-1);
interleukin-4 (IL-4);
tumor-necrotic factor-alpha (TNF-alpha).

No special preparation is required to take the test. The material for the study is a scraping from the buccal mucosa.

Treatment of schizophrenia

6. B-lymphocytes, development and differentiation. Function of B-lymphocytes, subpopulations of B-lymphocytes.

7. Methods for determining subpopulations of cells of the immune system. Flow cytometry to assess the subpopulation of lymphocytes.

8. Antigens: definition, properties, types.

9. Infectious antigens, types, characteristics.

10. Non-infectious antigens, types.

11. The hla-antigen system, role in immunology.

12. Immunoglobulins: definition, structure.

13. Classes of immunoglobulins, characteristics.

14. Antibodies: types, mechanisms of action. Monoclonal antibodies, production, use.

15. Serological reactions: general characteristics, purpose.

16. Precipitation reaction, reaction ingredients, purpose of formulation. Types of precipitation reaction (ring precipitation, diffusion in agar, immunoelectrophoresis). Methods for obtaining precipitating sera.

17. Dynamics of the immune response: nonspecific defense mechanisms.

18. Specific immune response to t-independent antibodies.

19. Specific immune response to T-dependent antibodies: presentation, processing, induction, effector phase

20.Immune response against intracellular microorganisms, tumor cells.

21.Mechanisms for limiting the immune response.

22. Primary and secondary immune response. Immunological tolerance.

23.Genetic control of the immune response.

24.Agglutination reaction: ingredients, its types, purpose.

25.RPG: ingredients, purpose. Coombs reaction: ingredients, purpose.

26. Neutralization reaction: types, ingredients, purpose.

27.Immune status, immunodiagnostic methods.

28. Characteristics of t- and b-lymphocytes, assessment methods. Cellular reactions: rbtl, rpml.

29. Characteristics of the system of granulocytes and monocytes. Assessment methods. Nst test. Characteristics of the complement system.

30. Reef: types, ingredients.

31. Ifa: ingredients, purpose of formulation, reaction accounting. Immunoblotting.

32.Ria: purpose of use, ingredients.

33.Vaccines, types, purpose of use.

34.Immune antisera and immunoglobulins.

35.Immunopotology. Classification. Main types. Immunotropic drugs.

36.Immunodeficiencies, types, causes.

37.Allergy: definition. General characteristics. Types of allergic reactions according to Gell-Coombs.

38. Immediate hypersensitivity reactions, types. Anaphylactic type of allergic reactions. Allergic diseases developing according to this mechanism.

39. Cytotoxic, immunocomplex, antireceptor reactions. Allergic and autoimmune diseases developing according to this mechanism.

40. Delayed hypersensitivity reactions. Allergic, autoimmune and infectious diseases developing according to this mechanism.

41. Autoimmune (autoallergic) diseases, classification. Mechanisms of development of certain autoimmune diseases.

42. Skin allergy tests, their use in diagnosis. Allergens for skin allergy tests, preparation, use.

43.Features of antitumor immunity. Features of immunity in the mother-fetus system

44.Natural immunity of the body to infectious diseases. "Hereditary immunity". Factors of natural innate immunity.

45. Humoral factors of nonspecific immunity.

46. Molecular images of pathogens and pattern recognition receptors. Toll-like receptor system.

47. Antigen-presenting cells, their functions.

48. System of mononucleon phagocytes, functions.

49.Phagocytosis: stages, mechanisms, types.

50. Granulocyte system, function.

51.Natural killer cells, activation mechanisms, function.

52. Complementary system: characteristics, activation pathways.

53.RSK: ingredients, mechanism, purpose.

3. Cytokines: general properties, classification. Interleukins.

Cytokines– these are peptide mediators secreted by activated cells that regulate interactions, activate all links of the SI itself and affect various organs and tissues. General properties cytokines: 1. They are glycoproteins. 2. Act on the cell itself and its immediate environment. These are short-distance molecules.3. They act in minimal concentrations. 4. Cytokines have specific receptors corresponding to them on the surface of cells 5. The mechanism of action of cytokines is to transmit a signal after interaction with the receptor from the cell membrane to its genetic apparatus. In this case, the expression of cellular proteins changes with a change in cell function (for example, other cytokines are released). Cytokines are divided into several main groups .1. Interleukins (IL)2. Interferons 3. Group of tumor necrosis factors (TNF) 4. Group of colony-stimulating factors (for example, granulocyte-macrophage colony-stimulating factor - GM-CSF) 5. Group of growth factors (endothelial growth factor, nerve growth factor, etc.) 6. Chemokines . Cytokines secreted primarily by cells of the immune system are called interleukins (ILs) - factors of interleukocyte interaction. They are numbered in order (IL-1 - IL-31). They are released by leukocytes when stimulated by microbial products and other antigens. IL-1 is secreted by macrophages and dendritic cells, causes an increase in temperature, stimulates and activates stem cells, T-lymphocytes, neutrophils, and is involved in the development of inflammation. It exists in two forms – IL-1a and IL-1b. IL-2 is secreted by T helper cells (mainly type 1, Th1) and stimulates the proliferation and differentiation of T and B lymphocytes, NK cells, and monocytes. IL-3 is one of the main hematopoietic factors, stimulates the proliferation and differentiation of early hematopoietic precursors, macrophages, and phagocytosis. IL-4 is a growth factor of B-lymphocytes, stimulates their proliferation at the early stage of differentiation; secreted by T-lymphocytes of the 2nd type and basophils. IL-5 stimulates the maturation of eosinophils, basophils and the synthesis of immunoglobulins by B-lymphocytes, produced by T-lymphocytes under the influence of antigens. IL-6 is a cytokine with multiple effects, secreted by T lymphocytes, macrophages and many cells outside the immune system, stimulates the maturation of B lymphocytes into plasma cells, the development of T cells and hematopoiesis, and activates inflammation. IL-7 is a lymphopoietic factor, activates the proliferation of lymphocyte precursors, stimulates the differentiation of T cells, is formed by stromal cells, as well as keratocytes, hepatocytes and other kidney cells. IL-8 is a regulator of the chemotaxis of neutrophils and T cells (chemokine); secreted by T cells, monocytes, endothelium. Activates neutrophils, causes their directed migration, adhesion, release of enzymes and reactive oxygen species, stimulates chemotaxis of T-lymphocytes, degranulation of basophils, adhesion of macrophages, angiogenesis. IL-10 - secreted by T lymphocytes (type 2 helper cells Th2 and regulatory T helper cells - Tr). Suppresses the release of pro-inflammatory cytokines (IL-1, IL-2, TNF, etc.) IL-11 - produced by bone marrow stromal cells, hematopoietic factor, acts similar to IL-3. IL-12 – source – monocytes-macrophages, dendritic cells causes proliferation of activated T-lymphocytes and natural killer cells, enhances the effect of IL-2. IL-13 – secreted by T lymphocytes, activates the differentiation of B cells. IL-18 – produced by monocytes and macrophages, dendritic cells, stimulates type 1 T helper cells and their production of interferon gamma, inhibits IgE synthesis.