Pathogenesis of tuberculosis

The development of tubercular inflammation depends on the reactivity of the body and the state of its protective forces, the virulence of mycobacteria tuberculosis and the duration of their persistence in the lungs. The action of various factors of the infectious process can explain a wide variety of tissue and cellular responses of the respiratory department, where specific changes are combined with nonspecific changes that somehow affect the manifestation and outcome of the main process.

Each stage is a complex complex of structural rearrangements of various body systems and respiratory organs, accompanied by deep changes in metabolic processes, the intensity of metabolic reactions of the respiratory department, is reflected in the morphofunctional state of its cellular and noncellular elements. It is important to study the earliest mechanisms of development of tuberculosis inflammation established in recent years.

[1], [2], [3], [4], [5], [6], [7], [8], [9], [10], [11]

Disturbances of microcirculation and condition of air-blood barrier

Already a day after intravenous injection of mycobacterium tuberculosis into the lung mice, the microcirculatory bed changes occur: it is possible to observe the widening of the profiles of the vascular capillary network, the erythrocyte sludging with the parietal arrangement of polymorphonuclear leukocytes. Electron microscopic analysis of the endothelial lining of the pulmonary capillaries shows activation of luminal cell surface, signs of intracellular edema development with disruption of micropinocytosis vesicles and their fusion into large vacuoles. Sites of the edematous, enlightened cytoplasm of endotheliocytes in places form sabotiform swelling that differ in quantity and size in different microvessels. In some cases, local exfoliation of their cytoplasmic processes from the underlying basal layer is observed, loosening and thickening of the latter.

Regardless of the mode of administration of mycobacterium tuberculosis, in all model experiments, an increase in the permeability of the air-blood barrier is observed in the first 3-5 days, as evidenced by the accumulation of fluid in the interstitium, the development of intracellular edema of not only endotheliocytes but also alveolocytes of type 1 (A1). Changes affect their cytoplasmic processes, in which appear areas of the enlightened, edematic cytoplasm, capable of bulging into the intra-alveolar space.

In the areas of generalization of mycobacterium tuberculosis and the development of pneumonic foci, the formation of primary granulomatous accumulations of mononuclears and polymorphonuclear leukocytes, A1 is determined with strongly thickened, locally destroyed cytoplasmic processes, sections of the exposed basal membrane. In many alveolocytes of type 2 (A2) swelling of apical microvilli occurs. Uneven expansion of mitochondrial profiles and the cytoplasmic network. Hyperhydration of the alveolar epithelium is sometimes accompanied by the release of fluid, plasma proteins and cellular elements of inflammation into the intra-vertex space.

Modern studies of microcirculation have made it possible to establish the leading role of the vascular system in the development of the initial phases of inflammation. Stimulated by cytokines, the endothelium secretes biologically active substances - adhesive molecules (selectin integrins). Various mediators (arachidonic acid metabolites) and growth factors, oxygen radicals, nitric oxide, etc., providing interaction between the endothelium and polymorphonuclear leukocytes, and between other cellular elements of inflammation. It has been established that L-selectin mediates the so-called "rolling neutrophil" effect. Is the initial stage of adhesion of these cells to the endothelium. Another species of selectin is P-selectin - after exposure to endothelial cells, histamine or oxygen metabolites is translocated to their surface, facilitating the adhesion of neutrophils. E-selectin is also detected on the surface of cytokine-activated endothelial cells; he is involved in the process of endothelium interaction of postcapillary venules with T-lymphocytes.

Cytokines. Isolated by mono- and polynuclei, cause structural rearrangement of the cytoskeleton of endothelial cells, which leads to their reduction and increase in capillary permeability. In turn, the passage of polymorphonuclear leukocytes through the wall of blood vessels can be accompanied by its damage and increased permeability for liquid and plasma proteins, and a change in the composition or activity of adhesive molecules leads to increased migration of monocytes and lymphocytes, which ensure the further development of the inflammatory reaction. Emerging in respiratory organs in response to the introduction of mycobacterium tuberculosis, it affects all structures of the respiratory department.

During the formation and maturation of tubercle granules, i.e. At the second stage of development of a specific process, disturbances in the structure of interalveolar septa grow. Edema, cell proliferation and fibrillogenesis in the interstitium significantly alter the morphofunctional state of the respiratory epithelium, especially near the foci of the inflammatory reaction. Violations of the conditions of the microenvironment and vital functions of alveolocytes adversely affect the functional state of the airgematic barrier and the gas exchange of the lungs.

Along with the already noted changes in the interalveolar septa in the edema zone, attention is drawn to the pronounced destructive changes in the alveolar epithelium, which can be traced on a considerable extent. They affect both types of alveolocytes and have one directionality - edema swelling of intracellular organelles, which leads to disruption of function, and then to cell death. Fragments of destroyed alveolocytes. Including A2, can be detected in the intra-alveolar contents. Macrophage elements, polymorphonuclear leukocytes, as well as a significant number of erythrocytes and eosinophils, reflecting the high permeability of the capillary network are located here. Among the destroyed cells determine the filaments of fibrin and their conglomerates.

In alveoli that retain air, it is also possible to observe signs of swelling of tissue and cellular structures of interalveolar septa. In addition, on the surface of the alveolar epithelium, bubble formation takes place, reflecting the initial stages of the destruction of the airgematic barrier and the "flooding" of the alveoli. At the final stage of development of tubercular inflammation, a progressive increase in the dystrophic and destructive changes in the structural components of the terminal lungs is observed, especially in the areas of the pulmonary parenchyma bordering the caseous necrotic foci or foci of tuberculosis pneumonia. Disturbances of the microcirculatory channel are widespread.

Transcapillary transition of blood plasma proteins contributes to the entry into the interstitium of light circulating immune complexes (CIC), which facilitate the development of both immunological and secondary immunopathological reactions in it. The role of the latter in the pathogenesis of tuberculosis is proven, and it is due to the intrapulmonary deposition of the CEC. A defect in the phagocyte system, an imbalance in the production of cytokines. Regulating intercellular interactions.

The area of the air lung parenchyma is reduced to 30% of the area of the cut, its areas alternate with areas of pronounced intra-alveolar edema, distelectase and atelectasis, emphysema enlargement of the alveoli. Despite the progressive nature of untreated tubercular inflammation, compensatory-regenerative processes take place in the pulmonary parenchyma free from foci. As our studies have shown, in the perifocal zone of inflammation, the functional activity of A2 is mainly aimed at maintaining the integrity of the alveolar epithelium, restoring the A1 population most sensitive to the action of the factors of the tuberculous process. The fact that A2 participates in the processes of regeneration as a cellular source of respiratory epithelium is now universally accepted. A significant increase in the proliferative activity of A2 in these zones is indicated by the detection of 6-10 nearby young alveolocytes, "growth buds" with the same type of well-developed nucleus structure, a significant content of mitochondria in the cytoplasm and polyribosomes, and a small number of secretory granules. Sometimes in these cells you can see the figures of mitosis. At the same time, intermediate-type alveolocytes, reflecting the transformation of A2 into A1, are extremely rare. Maintaining the gas exchange function of the organ is due to hypertrophy of the alveoli, the formation of growth points and the transformation of A2 in A1 in the remote areas of the pulmonary parenchyma. Here, the ultrastructural signs of the active secretory function of A2 are observed.

These data correlate with the results of electron microscopic examination of alveolar epithelium in the operating material. In patients with healing foci of tuberculosis infection, adenomatous structures are formed that resemble alveolar courses. The cells lining them have an ultrastructure of A2, which preserve single secretory granules. It is characteristic that the transformation of A2 into A1 does not occur (no alveolocytes of intermediate type are detected), which does not allow to relate these structures to newly formed alveoli, as some authors note.

The processes of restoring the respiratory epithelium, the formation of transition type alveolocytes, are observed only in the more distant pulmonary parenchyma, where nodular alveolocyte proliferation corresponding to the growth kidneys is determined. Here, the main gas exchange function of the lungs is realized, the cells of the air-blood barrier have a well-developed ultrastructure with a large number of micropinocytosis vesicles.

The study of various models of tubercular inflammation has shown that development in the lungs of specific inflammation is associated not only with certain destructive changes in the respiratory department directly in the foci of infection, but affects the entire pulmonary parenchyma, where signs of microcirculation disturbance are observed. Increase of permeability of blood vessels of interalveolar septa. With the progression of the inflammatory process, edema phenomena increase, which affects the condition of the alveolocytes, especially A1. The lumens of many alveoli are partially or completely filled with fluid and cellular elements of inflammation. Hypoxia and fibrotic changes of the interalveolar septa are reflected in the gas exchange function of the air-blood barrier, lead to the development of respiratory insufficiency and the death of experimental animals.

[12], [13], [14], [15]

The role of macrophages of the lungs

Macrophages of the lungs are a component of a single system for the whole organism of mononuclear phagocytes originating from the polypotent stem cell of the bone marrow. When dividing a stem cell, monocyte precursors, monoblasts and promonocytes, are produced. Monocytes circulate in the blood and partially exit into the interstitial tissue of the lungs, where for some time they may be in an inactive state. In the presence of inducers of differentiation, they are activated, move to the surface of the respiratory and bronchial epithelium, where several stages of maturation take place, transforming into alveolar and bronchial macrophages, respectively. The main function of these cells is the absorption function, which is related to their ability to phagocytosis a foreign material. Being one of the factors of natural resistance of the body, they protect those lung regions that first come into contact with microbes and abiogenic agents, i.e. Maintain the sterility of the epithelial lining of the lungs throughout its entire length. Most of the foreign material, as well as fragments of destroyed cell elements, are almost completely digested after conjugation of the phagosome vacuole of the macrophage (necrophage, hemosiderophage) with lysosomes containing proteolytic enzymes. For macrophages of the lungs, a high content of acid phosphatase, nonspecific esterase, cathepsins, phospholipase A2, and also enzymes of the Krebs cycle, especially succinate dehydrogenase, are characteristic. At the same time it is known that the pathogens of a number of infectious diseases, especially M. Tuberculosis, can persist for a long time in the cytoplasm of alveolar macrophages, since they have highly stable cell walls that oppose the action of lysosomal enzymes. In model experiments in untreated animals, in spite of the pronounced activation of acid phosphatase and other hydrolases, in the cytoplasm of alveolar macrophages it is possible to observe a certain proliferative activity of the mycobacterium tuberculosis and the formation of small colony-shaped aggregations by the pathogen.

The low microbicidal activity of lung macrophages is associated with organospecific features of phagocytes, since they function in a medium with a high oxygen content. The energy processes in their cytoplasm are mainly supported by the oxidative phosphorylation of lipoproteins, the catabolism of which is associated with one of the basic functions of these cells entering the pulmonary surfactant system. Extraction of energy, localization of oxidative processes affect the mitochondrial system, the development of which correlates with the functional state of the phagocyte. Here, too, superoxide dismutase is located, an antioxidant defense enzyme that catalyzes the dissmutation of singlet oxygen formed when electrons pass through the respiratory chain. This radically distinguishes macrophages from the lungs from polymorphonuclear leukocytes, which receive oxygen and bioenergy mainly due to glycolysis. In the latter case, the cleavage of the substrate occurs directly in the cytosol, and the activated oxygen and the hydrogen peroxide formed by myeloperoxidase constitute the main bactericidal potential for action on the bacteria.

Low biocidal macrophages of the lungs can be considered as a kind of payment for adaptation to aerobic conditions of functioning. Obviously, therefore, they carry out the fight against mycobacteria of tuberculosis together with polymorphonuclear leukocytes and monocytes of exudate (they are also called macrophages of inflammation). It is pathogenetically important that not all macrophages of the lungs that have captured mycobacterium tuberculosis are removed from the lungs with drift of the surfactant and bronchial secretion - some of them develop in the interstitium, which is the starting point for the formation of characteristic cell clusters - the granule.

Getting into the interstitium, rich in blood vessels, macrophages of the lungs with incomplete phagocytosis begin to produce inflammatory cytokines. Activating the adjacent endothelium. On the membranes of the latter, the expression of immunoglobulins increases, with the help of which selective adhesion of monocytes is carried out. Leaving the vascular bed, these cells are transformed into macrophages of exudate, which produce inflammatory mediators, which attract not only mono- but also polynuclears to the focus.

Simultaneously, the signal for the development of the granulomatous reaction comes from sensitized T-lymphocytes, the effectors of hypersensitivity of the delayed type, Among the lymphocytes. Which these cells begin to produce, the factor inhibiting the migration of monocytes, and IL-2, is of great importance for granulomeogenesis. They speed up the influx and fix the monocytes in the focus of infection, regulate their transformation into phagocytic, secreting and antigen presenting macrophages.

It is necessary to emphasize that. Being a mechanism of cellular respiratory protection against penetration of the pathogen, the granulomatous lung reaction in case of tubercular inflammation ultimately reflects the incompetence of mononuclear phagocytes in the fight against mycobacteria of tuberculosis. Therefore, macrophages are forced to continually proliferate (increase the number of populations) and differentiate into larger phagocytes (increase the quality of proteolysis). What are giant cells such as foreign bodies. In the phagosomes of the latter, not only the mycobacterium tuberculosis, but also large apoptotic cells, fragments of destroyed polymorphonuclear leukocytes can be seen under the electron microscope. In this case, the ultrastructural signs of proteolytic activity (the degree of development of the lysosomal apparatus) in such phagocytes per unit area of the cytoplasm do not significantly differ from the single-nuclear ones. In this regard, lung macrophages are constantly attracted to the focus of polymorphonuclear leukocytes, which have greater biocidal activity. Activation of the latter is accompanied by the release into the extracellular environment of a significant amount of hydrolases and oxidants, which leads to the disintegration of tissues. Formation of caseous masses in the center of the focus.

The most pronounced metabolic disturbances are observed in patients with acute progressive forms of pulmonary tuberculosis, which occur with predominance of exudative and alterative inflammatory reaction, and the course of progressive forms of pulmonary tuberculosis is characterized, as a rule, by expressed T-cell immunodepression. Suppression of T-cell immunity, pronounced lymphopenia lead to disruption of intercellular interactions, inhibition of granulomatous reaction.

Deficiency of activated monocytes and lymphocytes, combined with their morpho-functional insufficiency, may be a consequence of increased apoptosis. The imbalance of cytokines arising in such cases can serve as a marker of a defect in the immune system. The process of apoptosis has characteristic morphological features: chromatin condensation in the nuclear membrane, decay of the nucleolus, the formation of cellular fragments (apoptotic bodies) and their phagocytosis by macrophages.

With the peculiarities of the functioning of macrophages of the lungs, their ability not only to phagocytosis but also to the production of a large number of cytokines necessary for the activation and regulation of many extracellular reactions and processes occurring in the focus of tuberculous inflammation is associated. With their help self-regulation of the renewal and differentiation of mononuclears is carried out, intercellular interactions are built up under conditions of a specific process and regeneration.

Universal mediator of intercellular interactions is IL-1, the target for which are lymphocytes, polymorphonuclear leukocytes, fibroblasts. Endotheliocytes and other cellular elements. In this case, the secretory function of lung macrophages is built on the principles of self-regulation, when the same cell secures not only regulators of extracellular processes, but inhibitors that block their action. Secretory macrophages in their ultrastructural organization are significantly different from phagocytic. They rarely contain phagosome vacuoles and secondary lysosomes, but they have a developed vesicular apparatus and other ultrastructural signs of secretion. Especially well they are expressed in epithelioid cells, which belong to the hyperactive secretory macrophages.

Certain stages of differentiation of macrophages of the lungs can be clearly traced under the light and especially the electron microscope in the material of bronchoalveolar lavage. Depending on the structural organization of the nucleus and cytoplasm, young non-activated and biosynthetic mononuclears, as well as mature phagocytic and secreting macrophages, are identified among them. Young non-activated cells (15-18 microns in diameter) usually make up about 1/5 of all macrophage elements. They have a rounded nucleus with smooth contours: the cytoplasm is weakly basophilic, does not contain any inclusions. Under the electron microscope in these cells, rare profiles of the cytoplasmic network and mitochondria, several small lysosome-like granules, and free ribosomes are seen.

Activated, biosynthetic macrophages have larger sizes (18-25 microns in diameter), the nucleus differs in wavy contours and a distinct nucleolus. They have a basophilic cytoplasm, which contains developed long tubules of the granular cytoplasmic network and numerous polysomes. Elements of the lamellar complex are detected simultaneously in two or three zones, where primary lysosomes accumulate. Secondary lysosomes are represented by single inclusions; Phagosomes are rarely detected, which reflects the readiness of the cell to phagocytic function.

The diameter of mature macrophages of the lungs varies within wide limits (30-55 microns), which depends on the activity and functional orientation of the cells. The largest sizes are characteristic for macrophages with structural signs of pronounced phagocytosis. The surface of such cells forms numerous micro-growths and long pseudopodia. The oval or rounded nucleus is often located centrically, with wavy contours. A significant amount of condensed chromatin lies near the nuclear envelope, the nucleolus is shallow (1-1.2 μm). In the cytoplasm, inclusions, short tubules of the granular cytoplasmic network, cisterns and vacuoles of the plate complex, free ribosomes are determined. Cells contain a significant amount of mitochondria, primary (0.5-1 μm) and secondary (1.2-2 μm) lysosomes, as well as the phagosome vacuoles differing in size and number. The latter contain fragments of destroyed cell elements and mycobacterium tuberculosis ("necrophages", "hemosiderophages"), lamellar inclusions of phospholipid nature ("phospholiphages") and / or granules of neutral fat ("lipophags"), dust particles, tobacco tar, kaolin (" "," Smoker macrophages ").

In the presence of a permanent object of phagocytosis, multinucleated macrophages (more than 70 μm in diameter) appear with five or more nuclei. Typical cells of foreign bodies - the final stage of differentiation of the macrophage with phagocytic function - is determined in the granuloma and granulation tissue of tuberculosis foci. Macrophages of the lungs with pronounced secretory activity (25-40 microns in diameter) usually do not have typical pseudopodia. The nature of the surface can be compared with fine lacy ruggedness. Formed by numerous, relatively short micro-growths. A rounded or oval core contains a small amount of condensed chromatin, a clear large nucleolus (1.5-2 μm). Transparent cytoplasm practically does not contain large inclusions. Short channels of the granular cytoplasmic network are represented by single profiles, whereas well-developed elements of the lamellar complex are numerous vacuoles and vesicles with electronically transparent or osmiophilic contents. The same structures are detected in the ectoplasm, where they merge directly with the plasmolemma. Even in smokers with experience, in which all phagocytic cells contain characteristic inclusions of tobacco tar. Secreting macrophages have a small number of secondary lysosomes and single phagasm-like formations, i.e. Practically do not absorb the foreign material. Macrophages with ultrastructural signs of secretory activity under normal conditions are not more than 4-8% in broncho-alveolar lavage. Since the function of these cells is associated with the metabolism, synthesis and secretion of a variety of biologically active substances into the extracellular environment, any violations of the mechanisms of specific and non-specific defense lead to an increase in their number, the formation of macrophages with an increased secretory potential, the epithelioid cells. They form symplasts or, as a result of unfinished mitotic fission, turn into characteristic multinucleated cells of Pirogov-Langhans - a finale of macrophage differentiation with secretory activity.

Depending on the resistance of the organism, the nature of the action, the conditions of the microenvironment, the transformation processes of the growth of phagocytic, secretory or antigen presenting activity have their own peculiarities. It is shown that the calculation of the relative percentage in the bronchoalveolar lavage of morphofunctional types of macrophages (the definition of the macrophage formula) helps in the differential diagnosis of tuberculosis and other pulmonary granulomatosis, and allows to evaluate the effectiveness of the etiotropic treatment.

The ratio of the number of actively phagocytizing and synthesizing macrophages of the lungs not only reflects the nature of the tissue reaction in the tubercular inflammation zone, but can serve as an indicator of the activity of the pathological process. The problem of completeness of phagocytosis in tuberculosis also remains relevant. The results of our studies of the experimental and clinical material show that the outcome of the interaction between phagocytosis and the causative agent depends on the functional state of the macrophage and the biological properties of the microorganism.

Condition of the surfactant system

The achievements of the experimental-theoretical direction in the study of pulmonary surfactants allowed us to formulate a modern conception of the surfactant as a multicomponent system of cellular and noncellular elements, the structural and functional unity of which provides normal biomechanics of respiration.

To date, a certain amount of factual material has been accumulated, evidencing not only the significant adaptive capabilities of the surfactant system under conditions of profound restructuring of pulmonary ventilation and hemodynamics, but also the pronounced sensitivity of its components to many unfavorable factors of the tuberculosis process, the specific nature of which is determined by the duration of the persistence of the pathogen, the undulating course of the process , deep disturbances of the microcirculatory bed. The changes observed in this case affect not only the zones of formation of foci of infection, but also remote, actively functioning areas of the pulmonary parenchyma. In this regard, it is extremely important to assess the morpho-functional usefulness of various components of the surfactant system, to highlight those changes that can be used to diagnose surfactant-dependent disturbances of respiratory function and their timely correction.

The earliest signs of destruction of the pulmonary surfactant can be observed in model experiments using special methods of fixing the lung. At the initial stage of development of tubercular inflammation they are local in nature and are expressed mainly in the areas of intraalveolar edema. Under the electron microscope, it is possible to observe various stages of exfoliation and destruction of the outer film - the surfactant membrane by the edematous fluid. These changes fully manifest themselves in the focuses of tubercular inflammation, where the material of the destroyed surfactant is widely identified in the composition of intra-alveolar contents.

The noted changes in the extracellular lining of the alveoli take place in the foci of various bacterial pneumonia. In this case part A2. Primarily in perifocal alveoli, performs compensatory production of surface-active substances. A different picture is observed in the respiratory organs with the development of tubercular inflammation, since the pathogen has an adverse effect on the processes of intracellular synthesis of the surfactant. Direct introduction of mycobacterium tuberculosis into the lungs of dogs (puncture of the chest) showed that disorganization of the profiles of the cytoplasmic network and mitochondria is observed in A2 already in the first 15-30 minutes; After a few hours at the site of infection, the alveolocytes are completely destroyed. Rapid development of a deficiency of surfactants leads to a decrease in alveoli and the rapid spread of the inflammatory process to the surrounding parenchyma. In the adjacent alveolus, small young A2 with small small secretory granules or large cells with signs of vacuolization of intracellular structures, sometimes with completely destroyed cytoplasm, predominate. In those alveolocytes, where there are developed elements of the cytoplasmic network and lamellar complex, giant osmiophilic plate-like bodies (OPT) are revealed. Which indicates a delay (inhibition) removal of intracellular surfactant on the surface of the alveoli.

Mathematical modeling of the secretory function of A2 in the pulmonary parenchyma free from foci with increased functional load showed that despite the increase in the volume and density of mature secretory granules, the reserve potential of the population did not change significantly. It was found. That in conditions of increased vascular permeability, development of hypoxia and fibrotic changes of the interalveolar septa, the balance of the processes of deposition and maturation of the OPT in the direction of predominance of the latter is disturbed. Accelerated maturation of the OPT often results in an increase in the secretion granules of the electron-transparent matrix material, whereas the content of the osmiophilic surfactant material in them may be insignificant; The lamellar material of surfactants is loosely packed, occupying only 1 / 3-1 / 5 of the volume of the secretory granule. Violation of the initial stages of secretion can explain the appearance of a significant number of A2 with vacuolated OPT. Such cells usually have ultrastructural signs of destruction (enlightenment of the cytoplasmic matrix, edematous swelling of the mitochondria, tubules of the cytoplasmic network and lamellar complex), which indicates attenuation of the processes of intracellular production of the surfactant.

It is characteristic that a decrease in the synthesis of surface-active phospholipids is accompanied by the appearance in the cytoplasm of A2 granules of neutral lipids. Adequate reflection of lipid metabolism disorders in the lungs of experimental animals and humans affected by tuberculosis is the accumulation of macrophages-lipofagi (foamy cells) of varying maturity in the alveoli and the material of broncho-choleveolar lavage. In parallel, a significant increase in the lavage fluid content of neutral lipids and a decrease in the proportion of total phospholipids are observed.

One of the early signs of destruction of surfactant in the experiment and clinic of tuberculosis of respiratory organs is the loss of the ability of its membranes to form structures of reserve material. Instead, on the surface of the alveoli, in the phagosomes of alveolar macrophages and directly in the material of bronchoalveolar lavage, it is possible to see membranes ("giant layered spheres") twisted into tangles without a characteristic three-dimensional organization. The depth of the destructive changes in the surfactant system is also indicated by the detection frequency in the flush of the deflated A2. These data correlate with the results of biochemical and physico-chemical studies of pulmonary surfactants.

Taking into account all the revealed features, to characterize the state of the surfactant system, three degrees of its violations have been identified: minor, severe, widespread. The latter reflects an increased risk of development of surfactant-dependent respiratory failure in patients with advanced destructive forms of the disease.

The results of the research show that the processes that are associated with the increase of air-blood barrier permeability are the basis of the disturbances that arise in the surfactant lung system in tuberculosis:

• damage to the surfactant on the alveolar surface;
• change in metabolism and damage to A2;
• violation of removal mechanisms from the alveoli of the spent surfactant.

At the same time, studies have established that the main cytological mechanism supporting the functional potential of the surfactant system in altered tuberculosis inflammation is easy to increase the number of hypertrophic A2. Mainly in a distant from the specific focus of the pulmonary parenchyma.

[16], [17], [18], [19], [20], [21]

Genetic aspects of susceptibility to tuberculosis

Before we start the analysis of the current state of research in the field of mechanisms of antituberculous immunity and immunogenetics of tuberculosis, we consider it necessary to dwell on some common positions.

• First, mycobacteria, as is known, multiply and collapse mainly in macrophages. Very little data (and they are contradictory) suggests that. That there are some factors that can destroy the mycobacterium extracellularly.
• Second, there is no strong evidence that the neutrophilic phagocyte system plays a significant role in protecting against tuberculosis infection.
• Third, there is no strong evidence that antituberculosis antibodies can either destroy the mycobacterium extracellularly, or promote intracellular destruction in macrophages or some other type of cell.
• Fourth - there are a lot of facts supporting the clause about that. That the central link of anti-tuberculosis immunity is T-lymphocytes and that they exert their regulatory influence through the phagocyte system.
• Fifth - there is a number of evidence that hereditary factors play a significant role in tuberculosis infection.

Data that testify to the important role of genetic factors in susceptibility to tuberculosis in humans is convincing enough. First of all, this is indicated by the fact that with an extremely high infection rate of M. Tuberculosis (approximately one third of the adult population of the planet), the disease develops only in a small part of people. This is also indicated by a different level of susceptibility to infection in different ethnic groups and the inheritance of susceptibility and resistance to tuberculosis in families with multiple cases of the disease. Finally, the evidence of this situation is a significantly increased concordance of clinically expressed tuberculosis in monozygotic (identical) twins in comparison with dizygotic.

Traditional genetic studies in tuberculosis

The role of the main histocompatibility complex and NRAMP *

Identification of genes and their alleles, from the expression of which depends sensitivity or resistance to tuberculosis, would allow not only to penetrate deeply into the fundamental mechanisms of immunity and the development of the pathological process in tuberculosis, but would also bring to reality the use of genetic typing methods to identify among healthy people persons with genetically increased risk of tuberculosis infection, requiring priority prevention measures, in particular - a special approach to vaccination.

* - Natural resistance-associated macrophage protein is a macrophage protein associated with natural resistance.

There is a significant number of experimental studies showing the role of a number of genetic systems and individual genes (H2, BCG1, Tbc1, xid, etc.) in the resistance (sensitivity) to tuberculosis in mice. In humans, the genes of the main histocompatibility complex (MHC) of class II belong to the most studied ones, among which the HLA-DR2 (human) family allele complex shows a fairly high degree of association with increased morbidity in several ethnically distant populations, and the HLA-DQ locus alleles affect the clinical picture of tuberculosis. Recently, the first successes in the analysis of the connection with tuberculosis in people of the NRAMP1 gene have been achieved. These data are particularly noteworthy, since this gene has a high degree of homology with the NRAMP1 gene (selectively expressed in BCG 1 as it controls sensitivity to M. BovisBCG), which undoubtedly affects the susceptibility to intracellular pathogens (including including mycobacteria).

Mutations leading to loss of function

Several genes have been identified, with changes leading to a complete loss of the ability to code a functionally active product ("knockout" of the gene), especially the ability of mice to develop a protective immune response when infected with mycobacteria. These are the genes that encode IFN-γ. IL-12, TNF-α, as well as receptors of immune system cells to these cytokines. On the other hand, during the "knockout" of the genes encoding IL-4 and IL-10, the course of the tuberculosis infection did not differ much from that of the wild (original) type mice. These data confirmed the primary protective role of the immune system at the genetic level first of all, T1-lymphocytes) respond to infection by producing type 1 cytokines, but not type 2.

The applicability of these data to mycobacterial infections in humans was demonstrated. In very rare families in which children from a very early age suffered from relapsing mycobacterial infections and salmonellosis. The ultrahigh susceptibility is due to homozygous non-conservative mutations in genes that encode the receptors of cells to IFN-γ and IL-12, inherited from heterozygous for these mutations of parents; as was to be expected, with this inheritance of rare mutations, marriages were closely related. However, such gross violations lead to such high susceptibility to infections, which practically do not allow the child to survive for longer than several years. And even in almost sterile conditions.

These same considerations cause a somewhat skeptical assessment of the very approach of simulating animal infections with knockout mutations in genes that play a primary role in protecting against these infections. Such mutations lead to the expression of phenotypes that do not have a chance of surviving under normal conditions and would be quickly eliminated by selection. So. Mice that do not express MHC class II products and therefore do not have a normal pool of CD4 lymphocytes. After infection M. Tuberculosis in a short time die from disseminated infection. A very similar flow of tuberculosis in humans is observed with a pronounced drop in the number of CD4 cells in the late stages of AIDS. In solving the same issues of genetic determination of risk groups and in general for understanding the genetic causes of increased susceptibility within the normal population distribution, the researcher is concerned, although not with optimal (by this attribute), but quite viable individuals. This aspect of the problem speaks in favor of using more traditional experimental models for genetic analysis, for example, the interlinear differences in the flow of tuberculosis in mice.

Screening of the genome and previously unknown genes of susceptibility to tuberculosis

Back in the 1950s and 1960s, it was shown that the inheritance of signs of susceptibility and resistance to tuberculosis in laboratory animals is complex, polygenic. In this situation, first, it is necessary to choose clearly expressed, "extremely different" between sensitive and resistant animals or individuals phenotypes, that is, the characteristics of the disease, and then to investigate the nature of their inheritance. Secondly, it is necessary to take into account that a priori we have no idea about that. How many genes are involved in the control of the disease and how they are located in the genome. Therefore, it is necessary either to use genetic methods to reduce the genetic diversity in the studied population, which is split according to the studied feature (which is possible only in animal experiments), or to screen the whole genome using statistical methods not of Mendelian, but of quantitative genetics, or to combine these methods. After the methods of genome skinning were developed using PCR for microsatellite DNA sections and statistical processing and interpretation of the results, a genetic analysis of susceptibility to tuberculosis at a new level began.

The approaches mentioned above have recently been successfully applied in genetic experiments in linear mice by two groups of researchers. A group of authors from the Central Research Institute of the Russian Academy of Medical Sciences together with colleagues from the Center for the Study of Host Resistance at McGill University (Montreal, Canada) and the Royal Stockholm Institute were the first to genomic screening for inheritance in mice of the severity of the disease caused by intravenous administration of a high dose of M. Tuberculosis strain H37Rv. As lines of parents with opposite sensitivity to tuberculosis, the A / Sn (stable) and I / St (sensitive) lines were taken. A significant sensitivity coupling was found in females with at least three different loci located on chromosomes 3, 9 and 17. More recently, adhesion to loci in the proximal portion of chromosome 9 and the central portion of chromosome 17 has been shown for males. The locus of chromosome 9 was the most sensitive to sensitivity. Another group of researchers in the United States screened the mouse genome to determine the inheritance pattern of the M. Tuberculosa susceptibility trait of the Erdman strain. In a combination of C57BL / 6J mice (resistant in their model) and C3HeB / FeJ (sensitive) in the analysis of F2 hybrids. And then the descendants of BC1, the locus was mapped in the central part of chromosome 1. It controls the severity of the course of the disease. After primary mapping, a more accurate localization of the locus was achieved by recombination analysis, and its effect on such an important phenotypic trait as the severity of granulomatous pulmonary tissue damage was established in recurrent cross mice (BC3 generation), i.e. After the genetic diversity among the animals studied was significantly reduced through genetic techniques. It is important to note that the mapping locus. Received the designation sst1 (susceptibility to tuberculosis 1), although located in chromosome 1, certainly does not coincide with the locus NRAMP1. This is evidenced both by its localization on the chromosome, and the fact that the C57BL / 6 mice bear the allele of sensitivity to BCG for the NRAMP1 gene, but the resistance allele to M tuberculosis at the locus sst1.

Published in recent years, data on the presence of loci in the genome of the mouse, which fundamentally affect the character of the course of the tuberculosis process, allow us to hope for significant progress in this area and in analyzing the genetic susceptibility in humans. Fantastically rapid progress in genomic analysis is likely to make the transition from the genetics of mouse tuberculosis to genetics of human tuberculosis very fast, since the complete sequence of the genome of both humans and mice is practically deciphered.

Interaction of macrophage-mycobacterium

Macrophages play an extremely important role in protecting against tuberculosis infection both in the phase of antigen recognition and in the elimination of mycobacteria.

After penetration of mycobacteria into the lungs, the situation can develop in accordance with four main schemes:

• the primary reaction of the host may be sufficient to completely eliminate all mycobacteria, thereby eliminating the possibility of tuberculosis;
• in the case of rapid growth and multiplication of microorganisms, a disease known as primary tuberculosis develops;
• with latent infection, the disease does not develop, but mycobacteria persist in the body in the so-called resting state, and their presence is manifested only as a positive skin reaction to tuberculin;
• in some cases, mycobacteria are able to transition from a state of rest to a growth phase, and the latent infection is replaced by the reactivation of tuberculosis.

The first line of protection against infection, after the mycobacteria reached the lower part of the respiratory tract, is alveolar macrophages. These cells can directly suppress the growth of bacteria, phagocyting them. And also participate in a wide range of reactions of cellular antituberculous immunity - through the presentation of the antigen, stimulation of accumulation of T-lymphocytes in the inflammatory focus, etc. It is important to note that the specific mechanisms of binding of virulent and relatively avirulent strains of mycobacteria with phagocytes can differ.

There is sufficient evidence that the process of formation of vacuoles or phagosomes in the interaction of M. Tuberculosis with a mononuclear phagocyte is mediated by attachment of the microorganism to the complement receptors (CR1, CR3, CR4). Mannose receptors or other receptors of the cell surface. The interaction between the mannose receptors of phagocytic cells and mycobacteria is mediated, apparently, by the glycoprotein of the mycobacterial cell wall - lipoarabinomannan.

Cytokines of T-helper type 2-prostaglandin E2 and IL-4-stimulate the expression of CR and MP, and IFN-γ, on the contrary, inhibits the expression and function of these receptors, which leads to a decrease in the adhesion of mycobacteria to macrophages. Data on the participation in the attachment of bacteria to the receptor cells for proteins of the surfactant also continue to accumulate.

The role of the molecule CD14 (marker of phagocytes) was demonstrated in the model of interaction of mycobacteria with microglia-resident phagocytes of brain tissue. It has been established that antibodies to CD14 prevent infection of microglial cells with virulent laboratory strain H37Rv. Since the CD14 molecule does not permeate the cell membrane through and without therefore has direct contact with the cytoplasm, it is unable to transmit the lipoprotein-induced signal alone, but needs a co-receptor to activate intracellular signaling pathways. The most likely candidates for such co-receptors are representatives of the family of Toll-like receptors. Lipoproteins of microorganisms through the activation of these receptors on one hand can potentiate the protective mechanisms of the host organism, and on the other - through the induction of apoptosis lead to tissue damage. At the same time, apoptosis is able to inhibit the immune response by eliminating cells participating in immune responses, thereby reducing the damage to tissues.

In addition to the above, it seems likely that the so-called scavenger receptors play an important role in the process of attaching mycobacteria to phagocytic cells. Which are located on the surface of macrophages and have affinity for a number of ligands.

The fate of M. Tuberculosis after phagocytosis is suppression of its growth by macrophages. After entering the phagosome, pathogenic bacteria are affected by a number of factors aimed at their destruction. Such factors include the fusion of the phagosome with lysosomes, the synthesis of reactive oxygen radicals and the synthesis of reactive nitrogen radicals, in particular nitric oxide. The death of the mycobacterium within the macrophage can occur through several mechanisms as a result of complex, cytokine-mediated interactions between lymphocytes and phagocytes. It is possible that the ability of mycobacteria to avoid the toxic effects of reactive oxygen and nitrogen radicals is a key step in the transition to the latent stage of infection. The ability of the macrophage to inhibit the growth of M. Tuberculosis essentially depends on the stage of cell activation (at least in part) and on the balance of cytokines (primarily, probably, platelet-derived growth factor alpha (TGF-α) and IFN-γ).

An important component of the mechanism of antimycobacterial activity of macrophages is, apparently, apoptosis (programmed cell death). In the model of M.bovis BCG cultivation in monocytes it was shown that apoptosis (but not necrosis) of macrophages is accompanied by a decrease in the viability of phagocytized mycobacteria.

The role of T-lymphocytes in antituberculous immunity

T-lymphocytes are known to be the main component of acquired immunity in cases of tuberculosis infection. Immunization of experimental animals with mycobacterial antigens, as well as the course of tuberculosis infection, is accompanied by the generation of antigen-specific CD4 ⁺ and CD8 ⁺ lymphocytes .

The deficit of CD4 + and, to a lesser extent, CD8, observed in knockout mice according to the CD4, CD8, MHCII, MHCI, and antibodies specific for CD4 or CD8 antigens leads to a significant decrease in the resistance of mice to M. Tuberculosis infection. It is known that in AIDS patients, for whom CD4 ⁺ lymphocyte deficiency is characteristic , they note an extremely high sensitivity to tuberculosis. The relative contribution of CD4 ⁺ and CD8 ⁺ lymphocytes to a protective immune response can change at different stages of infection. Thus, in lung granulomas infected with M. BovisBCG, CD4 ⁺ T-lymphocytes predominate in the early stages of infection (2-3 weeks) . And at later stages the CD8 ⁺ lymphocyte count increases . With adoptive transfer, CD8 ⁺ lymphocytes , especially their subpopulation CD44 ^hl, have high proteomic activity. In addition to CD4 ⁺ and CD8 ⁺ lymphocytes , other lymphocyte subpopulations, in particular γδ and CD4 ⁺ CD8 ⁺ lymphocytes , are restricted to non-polymorphic MHC class CD1 molecules. Also, apparently, contribute to the protective immunity against tuberculosis infection. Mechanisms of effector action of T-lymphocytes are reduced mainly to the production of soluble factors (cytokines, chemokines) or to cytotoxicity. With mycobacterial infections, the predominant formation of T1 occurs, the characteristic features of which are the production of cytokines IFN-γ and TNF-α. Both cytokines are able to stimulate the antimycobacterial activity of macrophages than. In the first place, and the protective effect of CD4 lymphocytes is due. In addition, IFN-γ is able to suppress the severity of inflammatory reactions in the lungs and thereby reduce the severity of the course of tuberculosis infection. TNF-α is necessary for granuloma formation, full-fledged cooperation of macrophages and lymphocytes, and tissue protection from necrotic changes. Along with the protective effect, TNF-α has a "pathological" effect. Its products can lead to fever, loss of body weight and tissue damage - symptoms typical of tuberculosis infection. T-lymphocytes are not the only source of TNF-α. Its main producers are macrophages. The effect of TNF-α is largely determined by the level of production of other type 1 and 2 cytokines in the inflammatory focus. Under conditions of preferential production of type 1 cytokines and the absence of production of type 2 cytokines, TNF-α has a protective effect, while in the case of simultaneous production of type 1 and 2 cytokines, it is destructive. Since, as noted above, mycobacteria stimulate primarily T1 lymphocytes, the course of mycobacterial infections is usually not accompanied by an increase in production of IL-4 and IL-5. At the same time, with severe forms of infection, as well as in its late stages, there may be a local and systemic increase in the production of IL-4 and IL-5. Whether the increased production of type 2 cytokines is the cause of a more severe course of tuberculosis infection or its consequence is unclear.

Cytotoxicity to infected target cells has CD8 ⁺ cells as well as "nonclassical" CD8 ⁺ lymphocytes restricted by CDlb molecules, CD4 ⁺ CD8 ⁺ lymphocytes, and CD4 ⁺ lymphocytes . The value of cytotoxicity in protection in tuberculosis is indicated by a decrease in the cytotoxic activity of CD8 ⁺ lymphocytes and perforin content in patients with tuberculosis compared to healthy donors. Essential is the answer to the question of how the lysis of infected target cells can influence the course of the infectious process, whether it leads to a decrease in the intensity of reproduction of mycobacteria that are intracellular parasites, or vice versa, promotes the release of mycobacteria from infected macrophages and the infection of all new cells. The data of S. Stronger (1997). Seem to be able to contribute to an understanding of this problem. The authors have shown. That in cytotoxic lymphocytes there are granulinase molecules, which has a bactericidal effect on mycobacteria. To penetrate granulosin into infected cells, secretion of lymphocytes by proteins forming pores in the membrane of target cells is necessary. Thus, for the first time, data on the immediate destruction of mycobacteria (in macrophages) by T-lymphocytes were obtained and, thus, the possibility of direct involvement of T-lymphocytes in the protection of mycobacterial infections was demonstrated.

Regulation of the T-cell immune response

The response of T-lymphocytes and the production of effector cytokines by them are regulated by cytokines produced by antigen-presenting cells, including infected macrophages. IL-12 shifts the differentiation of T-lymphocytes towards the formation of Thl cells and stimulates the production of IFN-γ. Infection of IL-12 ^% M.bovis BCG mice leads to a progressive development of infection, increased dissemination of mycobacteria and is accompanied by a lack of granuloma formation in the lungs. In IL-12p40 ^{% of} infected M. Tuberculosis, uncontrolled growth of mycobacteria is associated with a violation of both natural resistance and acquired immunity and is caused by a significant decrease in the production of proinflammatory cytokines IFN-γ and TNF-β. Conversely, administration of recombinant IL-12 to mice and subsequent infection with M. Tuberculosis Erdmann results in an increase in their resistance to infection.

IL-10 is a regulatory cytokine that stimulates the development of humoral immunity reactions and suppresses many reactions of cellular immunity. It is believed that the effect of IL-10 on the T-cell response may be mediated by its effect on macrophages: IL-10 inhibits the presentation of macrophages by antigens and suppresses macrophage synthesis by pro-inflammatory cytokines TNF-α, IL-1, IL-6, IL-8 and IL -12, GM-CSF, G-CSF. IL-10 also has an anti-apoptotic effect. Such a spectrum of action, it would seem, should determine the significant effect of IL-10 on the intensity of antituberculous immunity, but the data on the dependence of protective immunity on IL-10 products are extremely controversial.

TGF-β is a unique factor of suppression of cellular immunity. The level of its production correlates with the severity of tuberculosis, and the introduction of mice infected with M. Tuberculosis, anti-TGF-β antibodies or natural inhibitors of TGF-β corrects a reduced T-cell response.

It should be noted that the effector role of T-lymphocytes is not limited to the production of cytokines and cellular cytotoxicity. Other processes occurring during the establishment of direct T-lymphocyte-macrophage contact, as well as the production of chemokines by T-lymphocytes, can make a significant contribution to the development of local inflammatory reactions. The latter, in turn, are due not only to the response of macrophages and T-lymphocytes. Neutrophils, eosinophils, fibroblasts, epithelial and other cells can be active participants in the processes occurring in the lungs in cases of tuberculosis infection.

Morphological studies of the process of granule formation, as well as the results of determining the dynamics of the formation of a specific T-cell response, allow us, in our opinion, to identify several stages of interaction of mycobacteria with a macroorganism. The first is characterized by a progressive multiplication of mycobacteria in the absence of a specific T-lymphocyte response and lasts about 2-3 weeks. The second occurs after the formation of mature T-lymphocytes and is characterized by stabilization of the growth of mycobacteria. As a rule, after this comes the stage of decompensation, coinciding in time with the destructurization of lymphoid formations and the appearance of necrotic changes in the lungs. The vaccine effect may be due to a reduction in the first phase of the response.