2 курс / Нормальная физиология / Учебное_пособие_по_физиологии_крови_Авдеева_Е_В_,_Репалова_Н_В_

Neutrophils are the first line of defense against microorganisms, especially bacteria. They are active phagocytes. Particles are taken up in a vacuole, the pH is lowered by a membrane proton pump, then the azurophilic granules fuse and empty. Killing and digestion occur. Activated oxygen species are also formed and kill microorganisms. Lysozyme destroys cell wall of gram-positive bacteria. The life span of neutrophil is only about 12-48 hours. Neutrophils undergo diapedisis and chemotaxis (fig. 18):

Diapedesis is the passage of metamorphosed WBCs through intact capillary walls into surrounding body tissue,

Chemotaxis is the attraction of neutrophils and macrophages to sites of actual or potential tissue trauma, mediated by the local release of trigger chemicals (metabolic production, antigen-antibody complex, bacteria, toxin, etc).

Fig. 18. Diapedesis and chemotaxis.

Phagocytosis is the process of ingestion and digestion by cells of solid substances, for example, other cells, bacteria, bits of necrotic tissue, foreign particles.

Fig. 19. Stages of phagocytosis.

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Phagocytosis can be non-specific but will be enhanced if the body has made specific antibodies due to previous exposure.

The number of neutrophil greatly increase occurring in acute inflammation and earlier time of chronic inflammation.

Eosinophils.

The function of eosinophils is not entirely clear, but their are increases in some parasitic infections, in allergic responses to a variety of stimuli including pollen, some drug reactions. Eosinophils are chemotoxic and kill parasites. Its number is lower in the morning and higher at night.

Fig. 20. Eosinophil.

Functions:

Detoxification and destruction of toxin of protein origin;

Destruction of antigen-antibody complexes;

Phagocytosis of basophil and mast cell granules that contain large amount of histamine;

They produce enzyme histaminase which destroys ingested histamine.

Basophils.

Although basophil possesses phagocytic capabilities, it is mainly the secretory cell which mediates the hypersensitivity reaction.

Fig. 21. Basophil.

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Functions:

Basophils (and mast cells) can bind IgE antibody, and, when subsequently exposed to the corresponding antigen, release vasoactive substances (heparin, histamine, chemotactic factors and chronic reactive material) leading to hypersensitivity reactions;

Histamine and chronic reactive material increase permeability of capillary and contract bronchia smooth muscle, and result in such allergic reaction as measles;

Heparin serves as lipase and speeds up fatty decomposition;

Eosinophil chemotactic factor A released by basophil can attract eosinophil collection and modify eosinophil function.

Circulatory time: 12 hours.

Monocytes.

Monocytes are the biggest of white blood cells (diameter about 15~30 µm) without granule, and are responsible for rallying the cells to defend the body. Monocytes carry out phagocytosis and are also called macrophages.

Fig. 22. Monocyte.

Functions:

It contains many nonspecific lipase and displays the powerful phagocytosis. They appear in the inflammation region after neutrophils and display the highest activity in acid medium.

As soon as monocytes get into tissue from blood , they change their name are called macrophages activating monocyte-macrophage system to release many cytokins, such as colony stimulating factor (CSF), IL-1, IL-3, IL-6, TNFα, INF-α, β, etc.

Cytokins induced by monocyte may modulate other cells growth.

Monocyte-macrophage system plays a very important role in specific immune responsive induction and regulation.

The monocyte-macrophage system includes promonocytes and their precursors in the bone marrow, monocytes in circulation and macrophages in tissues. After maturation in the bone marrow newly formed monocytes enter the circulation and migrate into different tissues; the half-life of monocytes in the blood stream is approximately three days. Once in the tissue monocytes undergo transformation into

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tissue macrophages with functional properties that are characteristic for the environment in which they reside. Macrophages play a central role in the immune regulation by presenting antigen to T-lymphocytes; they participate in ingestion and killing of various invading microorganisms. In addition, macrophages synthesize a great number of substances involved in host defense and inflammation i.e. complement components, prostaglandins, IL-1, tumor necrosis factor-alpha and others. During infection, macrophages have the capacity to become "activated" by lymphokines and different bacterial products; "activated" macrophages have an increased tumoricidal and microbicidal activity against various microorganisms, synthesis and secretion of immune mediators is enhanced. Monocyte-macrophage dysfunctions have been described in various disorders: defective chemotaxis (corticosteroids, drug induced immunosuppression, AIDS, diabetes), defective phagocytosis (lupus erythematosus, deficiency of membrane glycoprotein), microbicidal defect (chronic granulomatous disease), decreased cytotoxicity (Wiskott-Aldrich-Syndrome), deficiencies in the clearance of physiologic substrates in lysosomal diseases.

Lymphocytes.

Fig. 23. Lymphocyte.

Lymphocyte, type of white blood cell (leukocyte) that is of fundamental importance in the immune system because lymphocytes are the cells that determine the specificity of the immune response to infectious microorganisms and other foreign substances. In human adults lymphocytes make up roughly 20 to 40 percent of the total number of white blood cells. They are found in the circulation and also are concentrated in central lymphoid organs and tissues, such as the spleen, tonsils, and lymph nodes, where the initial immune response is likely to occur.

There are two main types of lymphocytes (fig. 24):

B cells,

T cells.

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mature largely in the bone marrow, in lymphoid tissue of gut, appendix and pharyngeal tonsils. All of the plasma cells descended from a single B cell produce the same antibody which is directed against the antigen that stimulated it to mature. The same principle concerns memory B cells. Thus, all of the plasma cells and memory cells "remember" the stimulus that led to their formation.

Function of В-lymphocytes

1.Immune memory.

2.Specific immunity. B-lymphocytes synthesize the immunoglobulins such as IgM, IgN, IgA, IgG, IgB, IgE.

T-cells destroy the body's own cells that have themselves been taken over by viruses or become cancerous.

Fig. 26. T-lymphocyte.

T-lymphocytes produced in the bone marrow and processed by the thymus and participate in cell-mediated immunity (such as graft rejection). There are different forms of T-lymphocytes (fig. 27):

T-killers or cytotoxic. These lymphocytes neutralize cells that carry antigen. One killer can neutralize one antigen-carrying cell. T-killers are able to destroy tumor cells, cells of nonself transplants, mutant-cells. They can form mediators of immunity

– lymphokines.

T-helpers. These cells distinguish antigen, interact with B-lymphocytes and help them to turn into plasmatic cells. Helpers’ products are interleukin-2, which helps in differentiation of additional T-cells and also the factor of B-cells growth that helps in differentiation of B-lymphocytes into plasmatic cells.

T-suppressors. These cells suppress overabundant activity of T- and B-lymphocytes, formation of antibodies, predicting in this way excess immune response. T-suppressors provide formation of immune tolerance (lack of immune response to self antigens and for the one, the organism already had contact with).

T-amplifiers. These cells activate T-killers. They regulate correlation between killers and suppressors.

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T-memory cells. These cells circulate in blood for years and after repeated contact, they distinguish antigen and provide secondary immune response, which is faster and more intensive.

Memory cells

Amplifiers

Fig. 27. Forms of T-lymphocytes.

Function of T-lymphocytes

1.Immune memory.

2.Anti-viruses immunity.

3.Anti-tissue immunity.

4.Regulate phagocytosis

Innate immunity refers to nonspecific defense mechanisms that come into play immediately or within hours of an antigen's appearance in the body. Innate (natural) immunity is so named because it is present at birth and does not have to be learned through exposure to an invader. These mechanisms include physical barriers such as skin, chemicals in the blood, and immune system cells that attack foreign cells in the body. The innate immune response is activated by chemical properties of the antigen. However, its components treat all foreign invaders in much the same way. They recognize only a limited number of identifying substances (antigens) on foreign invaders. However, these antigens are present on many different invaders. Innate immunity, unlike acquired immunity, has no memory of the encounters, does not remember specific foreign antigens, and does not provide any ongoing protection against future infection.

The white blood cells involved in innate immunity are : monocytes (which develop into macrophages), neutrophils, eosinophils, basophils, natural killer cells. Each type has a different function.

Acquired immunity obtained either from the development of antibodies in response to exposure to an antigen, as from vaccination or an attack of an infectious disease, or from the transmission of antibodies, as from mother to fetus through the placenta or the injection of antiserum. The acquired immune response is more

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complex than the innate. The antigen first must be processed and recognized. Once an antigen has been recognized, the immune system creates an army of immune cells specifically designed to attack that antigen. Acquired immunity also includes a "memory" that makes future responses against a specific antigen more efficient.

Acquired immunity can be active or passive. Active immunity results from the development of antibodies in response to an antigen, as from exposure to an infectious disease or through vaccination. Passive immunity results from the transmission of antibodies, as from mother to fetus through the placenta or by the injection of antiserum.

Fig. 28. The immune response.

RECOMENDED LABORATORY WORKS

1.Determination of osmotic resistance of erythrocytes.

2.Determination of erythrocyte sedimentation rate.

3.Calculation of leukocytes.

SUPPLEMENT

1. Osmotic resistence of erythrocytes.

The osmotic pressure inside the erythrocyte is a little higher than osmotic pressure of blood plasma. That’s why water is entering the erythrocyte, what provides

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normal turgor of the cell. In hypotonic solution, water moves inside the erythrocyte, resulting in swelling and rupture of the cell. It is called osmotic haemolysis.

For the work it is necessary to have: NaCl solutions (0.9%; 0.8%; 0.7%; 0.6%; 0.5%; 0.4%; 0.3%; 0.2%), 8 test-tubes, donor's blood.

The succession of the work. For the determination of the osmotic resistance of erythrocytes, the blood (1-2 drops) is placed into 8 test-tubes with equal quantities of sodium chloride solutions. The concentration of the solutions changes from 0.9% to 0.2% (step is 0.1%). The contents of the test-tubes is carefully mixed and left for an hour We distinguish 2 limits of osmotic resistance: minimum and maximum. The limit of minimum osmotic resistance is the concentration of the solution in which we observe the minimum hemolysis (fig. 29). In the case of minimum hemolysis we observe weak coloring of the solution by hemoglobin over the sediment (norm 0.48-0.42% solution). The limit of maximum osmotic resistance is the concentration of the solution in which we observe the maximum hemolysis and the formation of "uncelled" blood (norm 0.32-0.3% solution). No erythrocytes which resist hypotonic solution below 0.3% of NaCl.

Fig. 29. Osmotic resistence of erythrocytes.

The osmotic resistance of erythrocytes depends on the properties of erythrocytcs membrane and the cells shape. The presence of two limits of osmotic resistance of erythrocytes can be explained by the cells of different age which circulate in the blood. Old erythrocytes have less strongcover and less biconcave form than young eryithrocytes. During sickness (for example anemia) osmotic resistance is decreased and haemolysis occurs in higher concentrations of NaCl.

2. Determination of the ervthrocyte sedimentation rate (ESR).

Erythrocytes can not sedimentate inside blood vessels. Due to:

constant blood movement; the charge of blood vessel wall and the charge of erythrocyte is the same (negative) so cells repel from it.

If we put the blood inside the test-tube and add anticoagulant than in few minutes we can observe the sedimentation of erythrocytes, because the density of

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erythrocytes (1.090 g/cm3) is higher, than the density of blood plasma (1.025-1.034 g/cm3). The mechanism of the process is as follows. At first erythrocytes form complexes with each other (10-12 erythrocytes form “monetary column”). After these complexes interact with plasma proteins, they become heavier and they start to settle faster. Due to this process is not equable in time (slow in the beginning, faster in the end) ESR is determined for the fixed period of time, usually 1 hour.

For the work it is necessary to have: Panchenkov's apparatus, watch glass, 5% solution of sodium citrate (to prevent the blood coagulation), donor's blood.

Fig. 30. Panchenkov's apparatus: a – support of the Panchenkov's apparatus б – capillary.

The succession of the work. The capillary from Panchenkov's apparatus is washed by 5% solution of sodium citrate. Then the capillary is felt by the sodium citrate solution up to the mark "P" in the capillary and blow it out on the watch glass. Add (two times) the blood up to the "K" in the capillary. The blood is mixed with the solution of sodium citrate on the glass and is gathered up to the mark "O" in the capillary. Place the capillary in the support of the Panchenkov's apparatus for sedimentation during 1 hour. After 1 hour the blood is divided into plasma (above) and erythrocytes (below) (fig. 31).

In normal state ESR:

of men is 2-10 mm/hour,

of women is 2-15 mm/hour.

It depends of quantity of the blood cells (hematocrit), viscosity of the blood, protein composition of the blood, sex, and changes in disorders.

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