A. Interferons (IFN):

1. Natural IFN (1 generation):

2. Recombinant IFN (2nd generation):

a) short action:

IFN a2b: intron-A

IFN β: Avonex and others.

(pegylated IFN): peginterferon

B. Interferon inducers (interferonogens):

1. Synthetic- cycloferon, tiloron, dibazol and etc.

2. Natural- ridostin, etc.

V. Interleukins : recombinant interleukin-2 (roncoleukin, aldesleukin, proleukin, ) , recombinant interleukin 1-beta (betaleukin).

G. colony stimulating factors (molgramming, etc.)

Peptide preparations

Thymic peptide preparations .

Peptide compounds produced by the thymus gland stimulate the maturation of T-lymphocytes(thymopoietins).

With initially low levels, preparations of typical peptides increase the number of T-cells and their functional activity.

The founder of thymic preparations of the first generation in Russia was Taktivin, which is a complex of peptides extracted from the thymus of cattle. Thymic peptide complex preparations also include Timalin, Timoptin and others, and to those containing thymus extracts - Timimulin and Vilozen.

Preparations of peptides from bovine thymus thymalin, thystimulin administered intramuscularly and taktivin, timoptin- under the skin, mainly in case of insufficiency of cellular immunity:

With T-immunodeficiencies,

viral infections,

For the prevention of infections during radiation therapy and chemotherapy of tumors.

The clinical efficacy of first-generation thymic preparations is not in doubt, but they have one drawback: they are an undivided mixture of biologically active peptides that are rather difficult to standardize.

Progress in the field of drugs of thymic origin went along the line of creating drugs of the II and III generations - synthetic analogues of natural thymus hormones or fragments of these hormones with biological activity.

Modern drug Imunofan - hexapeptide, a synthetic analogue of the active center of thymopoietin, is used for immunodeficiencies, tumors. The drug stimulates the formation of IL-2 by immunocompetent cells, increases the sensitivity of lymphoid cells to this lymphokine, reduces the production of TNF (tumor necrosis factor), has a regulatory effect on the production of immune mediators (inflammation) and immunoglobulins.

Bone marrow peptide preparations

Myelopid obtained from a culture of bone marrow cells of mammals (calves, pigs). The mechanism of action of the drug is associated with stimulation of the proliferation and functional activity of B- and T-cells.

In the body, the target of this drug are B-lymphocytes. In violation of immuno- or hematopoiesis, the introduction of myelopide leads to an increase in the overall mitotic activity of bone marrow cells and the direction of their differentiation towards mature B-lymphocytes.

Myelopid is used in the complex therapy of secondary immunodeficiency states with a predominant lesion of the humoral immunity, for the prevention of infectious complications after surgical interventions, injuries, osteomyelitis, nonspecific pulmonary diseases, chronic pyoderma. Side effects of the drug are dizziness, weakness, nausea, hyperemia and soreness at the injection site.

All drugs in this group are contraindicated in pregnant women, myelopid and imunofan are contraindicated in the presence of Rhesus conflict between mother and fetus.

Immunoglobulin preparations

Human immunoglobulins

a) Immunoglobulins for intramuscular injection

Non-specific: normal human immunoglobulin

Specific: immunoglobulin against human hepatitis B, human antistaphylococcal immunoglobulin, human antitetanus immunoglobulin, human immunoglobulin against tick-borne encephalitis, human immunoglobulin against rabies virus, etc.

b) Immunoglobulins for intravenous administration

Non-specific: normal human immunoglobulin for intravenous administration (gabriglobin, immunovenin, intraglobin, humaglobin)

Specific: immunoglobulin against human hepatitis B (neohepatect), pentaglobin (contains antibacterial IgM, IgG, IgA), immunoglobulin against cytomegalovirus (cytotect), human immunoglobulin against tick-borne encephalitis, anti-rabies IG, etc.

c) Immunoglobulins for oral administration: immunoglobulin complex preparation (CIP) for enteral use in acute intestinal infections; anti-rotavirus immunoglobulin for oral administration.

Heterologous immunoglobulins:

anti-rabies immunoglobulin from horse serum, anti-gangrenous polyvalent horse serum, etc.

Preparations of nonspecific immunoglobulins are used for primary and secondary immunodeficiencies, preparations of specific immunoglobulins - for relevant infections (for therapeutic or prophylactic purposes).

Cytokines and preparations based on them

The regulation of the developed immune response is carried out by cytokines - complex complex of endogenous immunoregulatory molecules, which are the basis for creating a large group of both natural and recombinant immunomodulatory drugs.

Interferons (IFN):

1. Natural IFN (1 generation):

Alphaferons: human leukocyte IFN, etc.

Betaferons: human fibroblastic IFN, etc.

2. Recombinant IFN (2nd generation):

a) short action:

IFN a2a: reaferon, viferon, etc.

IFN a2b: intron-A

IFN β: Avonex and others.

b) prolonged action(pegylated IFN): peginterferon (IFN a2b + Polyethylene glycol), etc.

The main direction of action of IFN drugs is T-lymphocytes (natural killers and cytotoxic T-lymphocytes).

Natural interferons are obtained in a culture of donor blood leukocyte cells (in a culture of lymphoblastoid and other cells) under the influence of an inducer virus.

Recombinant interferons are obtained by a genetic engineering method - by cultivating bacterial strains containing in their genetic apparatus an integrated recombinant human interferon gene plasmid.

Interferons have antiviral, antitumor and immunomodulatory effects.

As antiviral agents, interferon preparations are most effective in the treatment of herpetic eye diseases (locally in the form of drops, subconjunctival), herpes simplex with localization on the skin, mucous membranes and genitals, herpes zoster (locally in the form of a hydrogel-based ointment), acute and chronic viral hepatitis B and C (parenterally, rectally in suppositories), in the treatment and prevention of influenza and SARS (intranasally in the form of drops). In HIV infection, recombinant interferon preparations normalize immunological parameters, reduce the severity of the disease in more than 50% of cases, cause a decrease in the level of viremia and the content of serum markers of the disease. In AIDS, combination therapy with azidothymidine is carried out.

The antitumor effect of interferon preparations is associated with an antiproliferative effect and stimulation of the activity of natural killers. IFN-alpha, IFN-alpha 2a, IFN-alpha-2b, IFN-alpha-n1, IFN-beta are used as antitumor agents.

IFN-beta-lb is used as an immunomodulator in multiple sclerosis.

Interferon preparations cause similar side effects. Characteristic - influenza-like syndrome; changes from the side of the central nervous system: dizziness, blurred vision, confusion, depression, insomnia, paresthesia, tremor. From the gastrointestinal tract: loss of appetite, nausea; on the part of the cardiovascular system, symptoms of heart failure are possible; from the urinary system - proteinuria; from the hemopoietic system - transient leukopenia. Rash, itching, alopecia, temporary impotence, nosebleeds may also occur.

Interferon inducers (interferonogens):

1. Synthetic - cycloferon, tiloron, poludan, etc.

2. Natural - ridostin, etc.

Interferon inductors are drugs that enhance the synthesis of endogenous interferon. These drugs have a number of advantages over recombinant interferons. They do not have antigenic activity. Stimulated synthesis of endogenous interferon does not cause hyperinterferonemia.

Tiloron(amiksin) refers to low molecular weight synthetic compounds, is an oral interferon inducer. It has a wide spectrum of antiviral activity against DNA and RNA viruses. As an antiviral and immunomodulatory agent, it is used for the prevention and treatment of influenza, SARS, hepatitis A, for the treatment of viral hepatitis, herpes simplex (including urogenital) and herpes zoster, in the complex therapy of chlamydial infections, neuroviral and infectious-allergic diseases, with secondary immunodeficiencies. The drug is well tolerated. Possible dyspepsia, short-term chills, increased overall tone, which does not require discontinuation of the drug.

Poludan is a biosynthetic polyribonucleotide complex of polyadenylic and polyuridylic acids (in equimolar ratios). The drug has a pronounced inhibitory effect on herpes simplex viruses. It is used in the form of eye drops and injections under the conjunctiva. The drug is prescribed for adults for the treatment of viral eye diseases: herpetic and adenovirus conjunctivitis, keratoconjunctivitis, keratitis and keratoiridocyclitis (keratouveitis), iridocyclitis, chorioretinitis, optic neuritis.

Side effects occur rarely and are manifested by the development of allergic reactions: itching and sensation of a foreign body in the eye.

Cycloferon- low molecular weight interferon inducer. It has antiviral, immunomodulatory and anti-inflammatory effects. Cycloferon is effective against tick-borne encephalitis, herpes, cytomegalovirus, HIV, etc. viruses. It has an antichlamydial effect. Effective in systemic connective tissue diseases. The radioprotective and anti-inflammatory effect of the drug was established.

Arbidol is prescribed orally for the prevention and treatment of influenza and other acute respiratory viral infections, as well as for herpetic diseases.

Interleukins:

recombinant IL-2 (aldesleukin, proleukin, roncoleukin ) , recombinant IL-1beta ( betaleykin).

Cytokine preparations of natural origin, containing a sufficiently large set of cytokines of inflammation and the first phase of the immune response, are characterized by a multifaceted effect on the human body. These drugs act on cells involved in inflammation, regeneration processes, and the immune response.

Aldesleukin- recombinant analogue of IL-2. It has an immunomodulatory and antitumor effect. Activates cellular immunity. Enhances the proliferation of T-lymphocytes and IL-2-dependent cell populations. Increases the cytotoxicity of lymphocytes and killer cells that recognize and destroy tumor cells. Enhances the production of interferon gamma, TNF, IL-1. Used for kidney cancer.

Betaleukin- recombinant human IL-1 beta. Stimulates leukopoiesis and immune defense. It is administered under the skin or intravenously in purulent processes with immunodeficiency, with leukopenia as a result of chemotherapy, with tumors.

Roncoleukin- a recombinant preparation of interleukin-2 - is administered intravenously for sepsis with immunodeficiency, as well as for kidney cancer.

Colony stimulating factors:

Molgramostim(Leikomax) is a recombinant preparation of human granulocyte-macrophage colony-stimulating factor. Stimulates leukopoiesis, has immunotropic activity. It enhances the proliferation and differentiation of precursors, increases the content of mature cells in the peripheral blood, the growth of granulocytes, monocytes, macrophages. Increases the functional activity of mature neutrophils, enhances phagocytosis and oxidative metabolism, providing mechanisms for phagocytosis, increases cytotoxicity against malignant cells.

Filgrastim(Neupogen) is a recombinant preparation of human granulocyte colony stimulating factor. Filgrastim regulates the production of neutrophils and their entry into the blood from the bone marrow.

Lenograstim- recombinant preparation of human granulocyte colony stimulating factor. It is a highly purified protein. It is an immunomodulator and a leukopoiesis stimulator.

Synthetic immunostimulants: levamisole, polyoxidonium isoprinosine, galavit.

Levamisole(decaris), an imidazole derivative, is used as an immunostimulant, as well as an antihelminthic agent for ascariasis. The immunostimulatory properties of levamisole are associated with an increase in the activity of macrophages and T-lymphocytes.

Levamisole is prescribed orally for recurrent herpetic infections, chronic viral hepatitis, autoimmune diseases (rheumatoid arthritis, systemic lupus erythematosus, Crohn's disease). The drug is also used for tumors of the large intestine after surgical, radiation or drug therapy of tumors.

Isoprinosine- a drug containing inosine. Stimulates the activity of macrophages, the production of interleukins, the proliferation of T-lymphocytes.

Assign inside for viral infections, chronic infections of the respiratory and urinary tract, immunodeficiencies.

Polyoxidonium- synthetic water-soluble polymer compound. The drug has an immunostimulating and detoxifying effect, increases the body's immune resistance against local and generalized infections. Polyoxidonium activates all factors of natural resistance: cells of the monocyte-macrophage system, neutrophils and natural killers, increasing their functional activity at initially reduced levels.

Galavit is a derivative of phthalhydrazide. The peculiarity of this drug is the presence of not only immunomodulatory, but also pronounced anti-inflammatory properties.

Drugs of other pharmacological classes with immunostimulating activity

1. Adaptogens and herbal preparations (phytopreparations): preparations of echinacea (immunal), eleutherococcus, ginseng, rhodiola rosea, etc.

2. Vitamins: ascorbic acid (vitamin C), tocopherol acetate (vitamin E), retinol acetate (vitamin A) (see section "Vitamins").

Echinacea preparations have immunostimulatory and anti-inflammatory properties. When taken orally, these drugs increase the phagocytic activity of macrophages and neutrophils, stimulate the production of interleukin-1, the activity of T-helpers, and the differentiation of B-lymphocytes.

Echinacea preparations are used for immunodeficiencies and chronic inflammatory diseases. In particular, immunal administered orally in drops for the prevention and treatment of acute respiratory infections, as well as together with antibacterial agents for infections of the skin, respiratory and urinary tract.

General principles for the use of immunostimulants in patients with secondary immunodeficiencies

The most reasonable use of immunostimulants seems to be in immunodeficiencies, manifested by increased infectious morbidity. Secondary immunodeficiencies remain the main target of immunostimulating drugs, which are manifested by frequent recurrent, difficult-to-treat infectious and inflammatory diseases of all localizations and any etiology. At the heart of each chronic infectious and inflammatory process are changes in the immune system, which are one of the reasons for the persistence of this process.

Immunomodulators are prescribed in complex therapy simultaneously with antibiotics, antifungal, antiprotozoal or antiviral agents.

· When carrying out immunorehabilitation measures, in particular in case of incomplete recovery after an acute infectious disease, immunomodulators can be used as monotherapy.

· It is advisable to use immunomodulators against the background of immunological monitoring, which should be carried out regardless of the presence or absence of initial changes in the immune system.

Immunomodulators acting on the phagocytic link of immunity can be prescribed to patients with both identified and undiagnosed immune status disorders, i.e. the basis for their use is the clinical picture.

A decrease in any parameter of immunity, revealed during an immunodiagnostic study in a practically healthy person, not necessarily is the basis for the appointment of immunomodulatory therapy.

Control questions:

1. What are immunostimulants, what are the indications for immunotherapy, what types of immunodeficiency states are divided into?

2. Classification of immunomodulators according to the preferential selectivity of action?

3. Immunostimulants of microbial origin and their synthetic analogues, their pharmacological properties, indications for use, contraindications, side effects?

4. Endogenous immunostimulants and their synthetic analogues, their pharmacological properties, indications for use, contraindications, side effects?

5. Preparations of thymic peptides and bone marrow peptides, their pharmacological properties, indications for use, contraindications, side effects?

6. Immunoglobulin preparations and interferons (IFN), their pharmacological properties, indications for use, contraindications, side effects?

7. Preparations of interferon inducers (interferonogens), their pharmacological properties, indications for use, contraindications, side effects?

8. Preparations of interleukins and colony-stimulating factors, their pharmacological properties, indications for use, contraindications, side effects?

9. Synthetic immunostimulants, their pharmacological properties, indications for use, contraindications, side effects?

10. Drugs of other pharmacological classes with immunostimulatory activity and general principles for the use of immunostimulants in patients with secondary immunodeficiencies?

Cytokines, by their nature, are proteins produced by cells of the immune system (often called "factors" in the literature). They are involved in the differentiation of newborn cells of the immune system, endowing them with certain features that are the source of diversity of immune cells, and also provide intercellular interaction. To make this process easier to understand, we can compare the production of immune cells 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, monocytes are obtained from identical cells.

Of interest to science is the peculiarity of the impact of a cytokine on a cell, which generates the production of other cytokines by this cell. That is, one cytokine triggers the production of others cytokines.

Cytokines, depending on the 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 a viral infection or other stimulus options. These proteins (cytokines) block the reproduction of the virus in other cells and take part in the immune intercellular 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:

• Enhance the production of interleukins (IL1)
• Lower the pH level in the intercellular environment with an increase in temperature
• Binds to healthy cells and protects them from viruses
• Able to inhibit cell proliferation (growth) by blocking the synthesis of amino acids
• Together 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. 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 as 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 clone, 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

To date, the following growth factors have been discovered:

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

The most studied are the functions of transforming growth factor beta. It is responsible for suppressing the growth and activity of T-lymphocytes, inhibits some functions of macrophages, neutrophils, B-lymphocytes. Although this factor refers to growth factors, in fact, it is involved in the reverse processes, 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 IgA immunoglobulin are enhanced during wound healing, and memory cells are generated.

Chemokines are low molecular weight cytokines. Their main function is to attract leukocytes from the bloodstream to the focus 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 (extreme exhaustion of the body as a result slows down the activity of the enzyme, which contributes to 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 (proliferation of blood vessels) and fibrogenesis (degeneration of tissue into connective tissue), granulomatosis (formation of granulomas - proliferation and transformation of phagocytes) and many other results.

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

First, we 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 immunity reactions, ensure their interconnection, 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 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 body systems. Interleukins are divided according to their functional activity into pro- and anti-inflammatory cytokines, growth factors of lymphocytes, 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, granulocytic CSF - G-CSF, macrophage-

ny KSF - M-CSF).

5. Chemokines- С, СС, СХС (IL-8), СХ3С - regulators of chemotaxis of various cell types.

6. Non-lymphoid cell growth factors- regulators of growth, differentiation and functional activity of cells of various tissue affiliations (fibroblast growth factor - FGF, endothelial cell growth factor, epidermal growth factor - epidermal EGF) 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. The secretion of cytokines is a short process. Cytokines do not persist as preformed molecules, but rather

synthesis always begins with the transcription of genes. Cells produce cytokines at low concentrations (picograms per milliliter).

4. In most cases, cytokines are produced and act on target cells that are 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 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 the reliability 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 the so-called decoy receptors. Soluble receptors are the extracellular domain of a membrane receptor separated by an enzyme. Soluble receptors are able to neutralize cytokines, participate in their transport to the focus of inflammation and in excretion from the body.

8. Cytokines work like a network. They can act in concert. Many of the functions originally attributed to a single cytokine appear to be due to the concerted action of several cytokines. (synergism 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 responses. The ability of some cytokines to increase or decrease 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 TNF-a 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 able to participate in the implementation of all of the above mechanisms.

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

From 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 a 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 system of regulation at the level of the whole organism, which ensures the development of protective reactions (for example, during infection).

In recent years, there has been an idea of ​​a cytokine system that combines:

1) producer cells;

2) soluble cytokines and their antagonists;

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

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

Let us first consider the main components of the cytokine system.

Cytokine producing cells

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

Rice. 7.1. Cytokine system

CD4 T-helpers 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 at very low concentrations.

Direction of differentiation Th0 determines the development of two forms of the 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 antigen capture and processing, 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 ]ChGK. IFNu provides differentiation of Th1, which begin to secrete cytokines (IL-2, IFNu, IL-3, TNFa, lymphotoxins), which regulate the development of reactions to intracellular pathogens

(delayed-type 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.), which determine the proliferation of B-lymphocytes, their further differentiation into plasma cells, and the development of antibody responses, mainly to extracellular pathogens.

IFNy 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 IFNy, directs T-cell differentiation 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 distinct subpopulation of T helper cells (Th17) producing IL-17 has been described. Members of the IL-17 family can be expressed by activated memory cells (CD4CD45RO), y5T cells, NKT cells, neutrophils, monocytes under the influence of IL-23, IL-6, TGFβ produced by macrophages and dendritic cells. ROR-C is the main differentiation factor in humans and ROR-γ in mice. 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 lymphocytes in the thymus can differentiate into natural regulatory cells (Treg) expressing CD4+ CD25+ surface markers and the FOXP3 transcription factor. These cells are able to suppress the immune response mediated by Th1 and Th2 cells through direct intercellular contact and synthesis of TGFβ and IL-10.

Schemes of differentiation of Th0 clones and the cytokines secreted by them are shown in Fig. 7.2 and 7.3 (see also color insert).

T-cytotoxic cells (CD8 +), natural killers - weak producers of cytokines, such as interferons, TNFa 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 manifestations of

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

Rice. 7.2. Different subpopulations of T-lymphocytes producing 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), e.g. repetitions, etc. As a result

This interaction with TLR triggers an intracellular signal transduction cascade leading to the expression of genes for two major groups of cytokines: pro-inflammatory and type 1 IFN (Fig. 7.4, see also color insert). Mostly 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 Th12 cells

III. Cells that are not part of the immune system (cells of the connective tissue, epithelium, endothelium) constitutively secrete autocrine growth factors (GGF, EGF, TGFr, etc.). and cytokines supporting 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 pro-inflammatory 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 can be soluble decoy receptors, antibodies, and the IL-1 receptor antagonist (IL-1RA). With the development of inflammation, there is an increase in the expression of the IL-1RA gene. But even normally, this antagonist is present in the blood at a high concentration (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 hematopoietic type of receptors, which have two extracellular domains, one of which contains a common sequence of amino acid residues of two tryptophan and serine repeats 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 IL-10 and IFN family receptors. The third type is represented by cytokine receptors belonging to the TNF group. The fourth type of cytokine receptor belongs to the superfamily of immunoglobulin receptors, which have extracellular domains similar in structure to those of immunoglobulin molecules. 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 are able to 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, with the help of a specific region of the TNF receptor family, the so-called "death" domain (Fig. 7.5, see color insert). Differentiation and activation 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.

The complex analysis of the cytokine system includes the following.

I. Evaluation of producer cells.

1. Expression definition:

Receptors that recognize a pathogen or TCR antigen, 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 transduction from the TNF receptor

Rice. 7.6. Jak-STAT - type 1 cytokine receptor 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. Evaluation of cytokines and their antagonists in the biological media 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. Target Cell Evaluation.

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

2. Determination of signal molecules in the intracellular content.

3. Determination of the functional activity of target cells.

Numerous methods for evaluating the cytokine system have been developed to provide diverse information. Among them are distinguished:

1) molecular biological methods;

2) methods for quantitative determination of cytokines using immunoassay;

3) testing the biological activity of cytokines;

4) intracellular staining of cytokines;

5) the ELISPOT method, which makes it possible to detect cytokines around a single cytokine-producing cell;

6) immunofluorescence.

We give a brief description of these methods.

Via molecular biological methods it is possible to study the expression of genes of cytokines, their receptors, signal molecules, to study the polymorphism of these genes. In recent years, a large number of studies have been performed that have revealed associations between allele variants of the cytokine system molecules genes 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 the real-time polymerase chain reaction - PCR-RT (see Chap. 6). hybridization method in situ allows you to clarify the tissue and cellular localization of the expression of cytokine genes.

The 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 the respective tissues after the extraction of tissue proteins or in natural fluids, such as tears, oral lavage, 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.

Determining the levels of cytokine production by peripheral blood mononuclear cells (PBMCs) shows the functional state of the cells. Spontaneous production of MNC cytokines in culture indicates that the cells are already activated. in vivo. Induced (by various stimulants, mitogens) cytokine synthesis 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 can serve as one of the signs of an immunodeficiency state. Cytokines are not specific for a particular antigen. Therefore, the specific diagnosis of infectious, autoimmune and allergic diseases by determining the level of certain cytokines is impossible. At the same time, the assessment of cytokine levels makes it possible to obtain data on the severity of the inflammatory process, its transition to the systemic level and prognosis, the functional activity of immune system cells, and 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 range of immunoassay methods, using polyclonal and monoclonal antibodies (see Chapter 4). ELISA allows you to find out what are the exact concentrations of cytokines in bio-

logical body fluids. ELISA cytokine detection has a number of advantages over other methods (high sensitivity, specificity, independence from the presence of antagonists, the possibility of accurate automated accounting, standardization of accounting). 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, their action on target cells. The study of the biological effects of cytokines has led to the development of four types of cytokine testing:

1) by induction of proliferation of target cells;

2) by cytotoxic effect;

3) by induction of differentiation of bone marrow progenitors;

4) by antiviral action.

IL-1 is determined by the stimulating effect on the proliferation of mouse thymocytes activated by a mitogen in vitro; IL-2 - according to the ability to stimulate the proliferative activity of lymphoblasts; for cytotoxic effects on mouse fibroblasts (L929), TNFa and lymphotoxins are tested. Colony stimulating factors are evaluated by their ability to support the growth of bone marrow progenitors as colonies on agar. The antiviral activity of IFN is detected by the inhibition of the cytopathic action 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. 7.1 is a list of cell lines used for cytokine testing. According to the ability to induce the proliferation of sensitive target cells, biotesting of IL-1, IL-2, IL-4, IL-6, IL-7, IL-15, etc. is carried out. However, these testing methods are not very sensitive and informative. Inhibitor and antagonist molecules can mask the biological activity of cytokines. Some cytokines exhibit general biological activity. Nevertheless, these methods are ideal for testing the specific activity of recombinant cytokines.

Table 7.1. Cell lines used to test the biological activity of cytokines

The end of the 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 body fluid. It is necessary to pay attention to a number of details.

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

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

Operating procedure

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

2. Prepare a series of consecutive two-fold dilutions of experimental (body fluids) and control samples. Biological fluids containing IL-1 or samples obtained by incubation of mononuclear cells without LPS and a laboratory standard preparation containing IL-1 are used as controls. In 96-well round bottom 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 completion of cultivation, 50 μl of a solution (1 μCi / ml) of [" 3 H]-thymidine are 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 harvester, the filters are dried and the inclusion of a label is determined by a liquid scintillation counter.

8. Results are expressed as stimulation coefficient.

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

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 for evaluating the functioning of a cytokine, but this method should be complemented by various types of appropriate controls for specificity using monoclonal antibodies. The addition of certain monoclonal antibodies to the cytokine in the culture blocks the biological activity of the cytokine, which proves that the signal for the proliferation of the cell line is the determined cytokine.

Using bioassay 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 reproduction of the test virus in cell culture.

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

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

Lab 7-2

Determination of interferon activity

1. A suspension of diploid human fetal fibroblasts on a medium with 10% serum of bovine embryos (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 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 test samples is carried out by the method of double dilutions on a monolayer of fibroblasts.

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

4. Wells with intact (untreated) virus-infected cells are used as controls.

Reference IFN samples with known activity are used as reference preparations in each study.

5. The sample dilution plates are incubated for 24 hours at 37°C in a 5% CO 2 atmosphere.

6. The level of IFN activity is determined by the reciprocal value 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. Antiserum cancels the action of the corresponding cytokine, which makes it possible to identify the type of IFN.

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

MYTH is considered as an important inflammatory mediator that activates 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 being accumulated about the role of MYTH in the pathogenesis of many inflammatory diseases, including sepsis, rheumatoid arthritis (RA), glomerulonephritis, etc. In RA, the concentration of MYTH 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.

There are various methods for testing the activity of MIF, when 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 action of MIF ( Suslov A.P., 1989).

Lab 7-3

Definition of MYTH activity

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

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

2. In the control wells add the culture medium (in 4 parallels) 100 µl.

3. A cell suspension of peritoneal macrophages is prepared, for which 2 hybrid mice (CBAxC57B1 / 6) F1 are intraperitoneally injected with 10 ml of Hank's solution with heparin (10 U / ml), the abdomen is gently massaged for 2-3 minutes. Then the animal is slaughtered by decapitation, the abdominal wall is carefully pierced in the groin area, and the exudate is aspirated through the needle with a syringe. Cells of peritoneal exudate are washed twice with Hank's 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. The MIGROSCRIN system is assembled, 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 an automatic pipette from Costar, USA (Fig. .7.7).

Insert the legs of the tripod into the corner wells of the plate. The cell suspension is collected with an automatic pipette into tips - 5 μl each, rinsed from excess cells by a single dipping 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 cells of the suspension settle to the bottom of the wells, where standard cell microcultures are formed.

5. Carefully remove the tip rack from the plate. The plate with microculture of cells is placed in a strictly horizontal position in a CO 2 incubator, where it is cultivated for 20 hours. During cultivation, the cells migrate along the bottom of the well.

6. Quantification of the results after incubation is carried out on a binocular loupe, visually assessing the size of the colony on a scale inside the eyepiece. Microcultures are shaped like a circle. The investigators then determine the average colony diameter from the results of colony measurements in 4 test or control wells. The measurement error is ±1 mm.

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

The sample has MYTH activity if the MI values ​​are equal to

For a conventional unit (U) of MYTH activity, the inverse value is taken equal to the value of the highest dilution of the sample (sample), at which the migration index is 0.6 ± 0.2.

The biological activity of PEOα is estimated by its cytotoxic effect on the line of transformed fibroblasts L-929. Recombinant TNFa is used as a positive control, and cells in a culture medium are used as a negative control.

The cytotoxic index (CI) is calculated:

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

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

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

For a conventional unit of TNF activity, the value of the reverse dilution of the sample is taken, which is necessary to obtain 50% cellular cytotoxicity. The specific activity of the sample is the ratio of activity in arbitrary units per 1 ml to the concentration of the protein contained in the sample.

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

The method of intracellular staining determines the expression of the cytokine at the level of one cell. Flow cytometry allows you to count the number of cells expressing a particular cytokine.

Let us list the main steps in the determination of intracellular cytokines.

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

The protein kinase C activator phorbol-12-myristate-13-acetate (PMA) in combination with the calcium ionophore ionomycin (IN) is most often used as a cytokine inducer. The use of this combination causes the synthesis of a wide range of cytokines: IFNu, IL-4, IL-2, TNFα. The disadvantage of using FMA-IN is the problem of detecting 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, incubate for 10-15 min 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 cytokines to be determined. Preliminary staining of surface markers (CD4, CD8) increases the amount of information obtained about a 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 the population, the limiting dilution method and the ELISPOT variant of the enzyme-linked immunosorbent assay (see Chapter 4) are used.

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 of the organ and preparation of 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 the threshold concentrations of the secreted product. However, they do not determine the amount of secreted cytokine and can be technically complex. A variety of careful monitoring for non-specific reactions is necessary.

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

Thus, the assessment of the cytokine system is extremely important for characterizing the state of the body's immune system. The study of different levels of the cytokine system makes it possible 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 cytokine producing cells.

5. Describe the families of cytokine receptors.

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

7. Tell us about the production of cytokines in the innate immune system.

8. What are the main approaches to the complex 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 of 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 detect a defect at the level of the cytokine receptor.

15. Describe the sequence of methods used to detect 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?

METHODS FOR THE DETERMINATION OF CYTOKINES

S.V. Sennikov, A.N. Silkov

The review is devoted to the main methods for studying cytokines currently used. The possibilities and purpose of the methods are briefly characterized. The advantages and disadvantages of various approaches to the analysis of cytokine gene expression at the level of nucleic acids and at the level of protein production are presented. (Cytokines and inflammation. 2005. V. 4, No. 1. S. 22-27.)

Keywords: review, cytokines, methods of determination.

Introduction

Cytokines are regulatory proteins that form a universal network of mediators, characteristic of both the immune system and cells of other organs and tissues. Under the control of this class of regulatory proteins, all cellular events occur: proliferation, differentiation, apoptosis, and specialized functional activity of cells. The effects of each cytokine on cells are characterized by pleiotropy, the range of effects of different mediators overlaps, and, in general, the final functional state of the cell depends on the influence of several cytokines acting synergistically. Thus, the cytokine system is a universal, polymorphic regulatory network of mediators designed to control the processes of proliferation, differentiation, apoptosis, and functional activity of cellular elements in the hematopoietic, immune, and other homeostatic systems of the body.

Little time has passed since the description of the first cytokines. However, their research led to the allocation of an extensive section of knowledge - cytokinology, which is an integral part of various fields of knowledge and, first of all, immunology, which gave a powerful impetus to the study of these mediators. Cytokinology permeates all clinical disciplines, ranging from the etiology and pathogenesis of diseases to the prevention and treatment of various pathological conditions. Therefore, researchers and clinicians need to navigate the diversity of regulatory molecules and have a clear understanding of the role of each of the cytokines in the processes under study.

Methods for the determination of cytokines over 20 years of their intensive study have undergone a very rapid evolution and today represent a whole area of ​​scientific knowledge. At the beginning of the work, researchers in cytokinology are faced with the question of choosing a method. And here the researcher must know exactly what information he needs to obtain in order to achieve his goal. Currently, hundreds of different methods for assessing the cytokine system have been developed, which provide diverse information about this system. Cytokines can be assessed in various biological media by their specific biological activity. They can be quantified using a variety of immunoassay methods using poly- and monoclonal antibodies. In addition to studying the secretory forms of cytokines, one can study their intracellular content and production in tissues by flow cytometry, Western blotting, and in situ immunohistochemistry. Very important information can be obtained by studying cytokine mRNA expression, mRNA stability, the presence of cytokine mRNA isoforms, and natural antisense nucleotide sequences. The study of allelic variants of cytokine genes can provide important information about the genetically programmed high or low production of a particular mediator. Each method has its own advantages and disadvantages, its own resolution and accuracy of determination. Ignorance and misunderstanding of these nuances by the researcher can lead him to false conclusions.

Determination of the biological activity of cytokines

The history of the discovery and the first steps in the study of cytokines was closely associated with the cultivation of immunocompetent cells and cell lines. Then the regulatory effects (biological activity) of a number of soluble protein factors on the proliferative activity of lymphocytes, on the synthesis of immunoglobulins, and on the development of immune responses in in vitro models were shown. One of the first methods for determining the biological activity of mediators is the determination of the human lymphocyte migration factor and its inhibition factor. As the biological effects of cytokines were studied, various methods for assessing their biological activity also appeared. So, IL-1 was determined by assessing the proliferation of mouse thymocytes in vitro, IL-2 - by the ability to stimulate the proliferative activity of lymphoblasts, IL-3 - by the growth of hematopoietic colonies in vitro, IL-4 - by the comitogenic effect, by increasing the expression of Ia proteins , by inducing the formation of IgG1 and IgE, etc. . The list of these methods can be continued, it is constantly updated as new biological activities of soluble factors are discovered. Their main drawback is the non-standard methods, the impossibility of their unification. Further development of methods for determining the biological activity of cytokines led to the creation of a large number of cell lines sensitive to one or another cytokine, or multisensitive lines. Most of these cytokine-responsive cells can now be found on lists of commercially available cell lines. For example, for testing IL-1a and b, the D10S cell line is used, for IL-2 and IL-15, the CTLL-2 cell line is used, for IL-3, IL-4, IL-5, IL-9, IL-13, GM-CSF - cell line TF-1, for IL-6 - cell line B9, for IL-7 - cell line 2E8, for TNFa and TNFb - cell line L929, for IFNg - cell line WiDr, for IL-18 - cell line line KG-1.

However, such an approach to the study of immunoactive proteins, along with well-known advantages, such as measuring the real biological activity of mature and active proteins, high reproducibility under standardized conditions, has its drawbacks. These include, first of all, the sensitivity of cell lines not to one cytokine, but to several related cytokines, the biological effects of which overlap. In addition, the possibility of inducing the production of other cytokines by target cells, which can distort the test parameter (as a rule, these are proliferation, cytotoxicity, chemotaxis), cannot be ruled out. We do not yet know all the cytokines and not all of their effects, so we evaluate not the cytokine itself, but the total specific biological activity. Thus, the assessment of biological activity as the total activity of different mediators (insufficient specificity) is one of the disadvantages of this method. In addition, using cytokine-sensitive lines, it is not possible to detect non-activated molecules and associated proteins. This means that such methods do not reflect the real production for a number of cytokines. Another important disadvantage of using cell lines is the need for a cell culture laboratory. In addition, all procedures for growing cells and incubating them with the studied proteins and media require a lot of time. It should also be noted that long-term use of cell lines requires renewal or re-certification, since as a result of cultivation they can mutate and be modified, which can lead to a change in their sensitivity to mediators and a decrease in the accuracy of determining biological activity. However, this method is ideal for testing the specific biological activity of recombinant mediators.

Quantification of cytokines using antibodies

Cytokines produced by immunocompetent and other cell types are released into the intercellular space for paracrine and autocrine signaling interactions. By the concentration of these proteins in the blood serum or in a conditioned environment, one can judge the nature of the pathological process and the excess or deficiency of certain cell functions in a patient.

Methods for determining cytokines using specific antibodies are currently the most common detection systems for these proteins. These methods went through a whole series of modifications using different labels (radioisotope, fluorescent, electrochemiluminescent, enzymatic, etc.). If radioisotope methods have a number of disadvantages associated with the use of a radioactive label and the limited time of using labeled reagents (half-life), then enzyme immunoassay methods are the most widely used. They are based on the visualization of insoluble products of an enzymatic reaction that absorb light of a known wavelength in quantities equivalent to the concentration of the analyte. Antibodies coated on a solid polymer base are used to bind the substances to be measured, and for imaging, antibodies conjugated to enzymes, typically alkaline phosphatase or horseradish peroxidase.

The advantages of the method are obvious: it is a high accuracy of determination under standardized conditions for storing reagents and performing procedures, quantitative analysis, and reproducibility. The disadvantages include the limited range of determined concentrations, as a result of which all concentrations exceeding a certain threshold are considered equal to it. It should be noted that the time required to complete the method varies depending on the manufacturer's recommendations. However, in any case, we are talking about several hours required for incubation and washing of reagents. In addition, latent and bound forms of cytokines are determined, which in their concentration can significantly exceed free forms, mainly responsible for the biological activity of the mediator. Therefore, it is desirable to use this method together with methods for assessing the biological activity of the mediator.

Another modification of the immunoassay method, which has found wide application, is the electrochemiluminescent method (ECL) for the determination of proteins with antibodies labeled with ruthenium and biotin. This method has the following advantages compared to radioisotope and enzyme immunoassays: ease of implementation, short execution time of the technique, no washing procedures, small sample volume, large range of determined cytokine concentrations in serum and in a conditioned medium, high sensitivity of the method and its reproducibility. The considered method is acceptable for use both in scientific research and in clinical.

The following method for evaluating cytokines in biological media is based on flow fluorometry technology. It allows you to simultaneously evaluate up to a hundred proteins in a sample. Currently, commercial kits have been created for the determination of up to 17 cytokines. However, the advantages of this method also determine its disadvantages. Firstly, this is the laboriousness of selecting optimal conditions for the determination of several proteins, and secondly, the production of cytokines is cascaded with production peaks at different times. Therefore, the determination of a large number of proteins at the same time is not always informative.

The general requirement of immunoassay methods using the so-called. "sandwich", is the careful selection of a pair of antibodies, which allows you to determine either the free or bound form of the analyzed protein, which imposes limitations on this method, and which must always be taken into account when interpreting the data obtained. These methods determine the total production of cytokines by different cells, while at the same time, antigen-specific production of cytokines by immunocompetent cells can only be judged tentatively.

Currently, the ELISpot (Enzyme-Liked ImmunoSpot) system has been developed, which largely eliminates these shortcomings. The method allows semi-quantitative assessment of cytokine production at the level of individual cells. The high resolution of this method makes it possible to assess antigen-stimulated cytokine production, which is very important for assessing a specific immune response.

The next, widely used for scientific purposes, method is the intracellular determination of cytokines by flow cytometry. Its advantages are obvious. We can phenotypically characterize a population of cytokine-producing cells and/or determine the spectrum of cytokines produced by individual cells, and it is possible to characterize this production relatively. However, the described method is rather complicated and requires expensive equipment.

The next series of methods, which are used mainly for scientific purposes, are immunohistochemical methods using labeled monoclonal antibodies. The advantages are obvious - determining the production of cytokines directly in tissues (in situ), where various immunological reactions occur. However, the methods under consideration are very laborious and do not provide accurate quantitative data.

Cytokines include a variety of proteins with a molecular weight of 15-40 kDa, which are synthesized by various cells in the body. Cytokines are molecules that ensure the interaction of cells of the immune system, vascular endothelium, nervous system, and liver. Currently, more than 200 cytokines are known.

The same cytokines can be synthesized by cells of different types - the immune system, spleen, thymus, connective tissue. On the other hand, a particular cell is capable of producing many different cytokines. The greatest variety of cytokines is formed by lymphocytes, due to this, lymphocytic immunity interacts with other immune mechanisms and with the body as a whole.

An essential feature of cytokines, unlike hormones and other signaling molecules, is the same, different, or even opposite result of their action for different cells. Those. The final result of the impact of a cytokine does not depend on its type, but on the internal program of the target cell, on its individual tasks!

Functions of cytokines

The role of cytokines in the regulation of body functions can be divided into 4 main components:

1. Regulation of embryogenesis, laying and development of organs, including organs of the immune system.

2. Regulation of tissue growth processes:

3. Regulation of individual physiological functions:

• ensuring the functional activity of cells,
• coordination of reactions of the endocrine, immune and nervous systems,
• maintaining homeostasis (dynamic constancy) of the body.

4. Regulation of protective reactions of the body at the local and systemic level:

• changes in the duration and intensity of immune responses (antitumor and antiviral protection of the body),
• modulation of inflammatory responses,
• participation in the development of autoimmune reactions.
• stimulation or inhibition of cell growth,
• participation in the process of hematopoiesis.