Reactions of the Immune and Hematopoietic Systems of Mice with Different Genotypes to Stress

The dissertation is devoted to the study of the peculiarities of reactions of the immune and hematopoietic systems of mice with different genotypes to the administration of a thymus-dependent antigen and immobilization stress, as well as the development of approaches to correction of the arising violations using natural origin preparations. Five inbred mouse lines differing in reactivity to the antigenic stimulus were used in the work. The state of the hematopoietic and lymphoid tissue of the bone marrow, thymus, and spleen was investigated, as well as indicators of humoral immunity and nonspecific resistance of the organism. Differences in the reaction of high-responding and low-responding lines to the combined impact of the antigen and stress were revealed. Correction of immunodeficient states was conducted using extracts of Altai hellebore, pantocrin, thick-leaved rhodiola rosea, ivy-leaved birdfoot trefoil, creeping peony, and purple coneflower tincture.

Relevance.

The rapid development of human technogenic activity, intensive environmental pollution, and other extraordinary-impact circumstances create quite harsh external conditions for vital activity that are inadequate to the innate and acquired properties of the organism. Staying in such conditions leads to additional expenditure of the reserve capacities of homeostatic systems [Meerson F.Z., Pshennikov M.G., 1988; Novikov B.C., Smirnov B.C., 1995; Goldberg E.D., Dygai A.M., Uduut V.V. et al., 1996; Goldberg E.D., Dygai A.M., Khlusov I.A., 1997; Goldberg E.D., Dygai A.M., Zhdanov V.V., Goldberg V.E., 2001; Goldberg E.D., Dygai A.M., Suslov N.I. et al., 2003; Masnaya N.V., Churin A.A., Borsuk O.S. et al., 2003; Goldberg E.D., Dygai A.M., Provalova N.V. et al., 2004].

Research of recent years has shown that the organism's reaction to stress is accompanied by depression of the functional activity of various organ systems [Kort W.J., 1994; Nestorova I.V., 1999; McCarty R., Aguilera G., Sabban E.L., Kvetnansky R., 2002; Goldberg E.D., Dygai A.M., Provalova N.V. et al., 2004]. One of the leading places in these processes belongs to the immune and hematopoietic systems. On the one hand, these are effector systems performing homeostatic functions, similar to the digestive, endocrine, and others. On the other hand, the immune and hematopoietic systems perform, similar to the neuroendocrine system, regulatory functions with respect to proliferation and differentiation of cells, possibly, of all tissues of the organism [Kort W.J., 1994; Abramov V.V., Abramova T.Ya., 1996; Kozlov V.A., 1997; Abramov V.V., Egorov D.N., Vardosanzidze K.V., Kozlov V.A., 1998; Devoyno L.V., 1998; Shirinsky I.V., Shirinsky B.C., 2001; Fullford A.J., Jessop D.S., 2002]. Hence arise early changes in the immune and hematopoietic systems occurring already at the initial stages of adaptive reactions, and "pervasive" participation of the immune system in the pathogenesis of many human diseases.

In case of exhaustion of the adaptive mechanisms, a complex of changes classified as a secondary immunodeficient state is formed, which is the most expected reaction of the organism to any extraordinary impact and one of the etiological causes of development of immunodependent diseases [Novikov B.C., Smirnov B.C., 1995; Kovalchuk Yu.B., Pinegin B.V., 1999; Lolor G., Fischer T., Adelmann D., 2000; Nestorova I.V., 1999, 2002].

However, despite the unceasing interest of researchers and physicians to this problem worldwide, the question of the mechanism of development and the nature of changes in the immune and hematopoietic systems in response to extraordinary impact remains open. In most cases, only diagnostic detection of manifestations of the immunopathogenesis is possible to confirm the diagnosis of this or that immunodeficiency, and only discussion of the etiological aspects of these states. This is especially relevant in clinical immunology, where the question of the etiology of many primary and secondary immunodeficient states remains unresolved [Novikov B.C., Smirnov B.C., 1995; Kozlov V.A., 1997; Lolor G., Fischer T., Adelmann D., 2000; Nestorova I.V., 1999, 2002].

The search for a solution to the problem of correction of the mentioned immune system states led to the appearance in the mid-1980s of a new direction in medicine — immunorehabilitation [Petrov R.V., 1984; Novikov B.C., Smirnov B.C., 1995; Sepiashvili R.I., 1996, 1999; Karaulov A.B., 1999; Nestorova I.V., Starchenko A.A., Ivanova S.A., Simbirtsev A.S., 2002; Starchenko A.A., 2002] and the emergence of new pharmaceutical preparations with immunotropic action. The list of preparations is quite large; it includes agents possessing not only immunostimulating, modulatory, corrective, and suppressive actions, but also passive replacement therapy agents, to which belong both immunoglobulins and, in some cases, thymic factors, interleukins, interferons, colony-stimulating factors, and other cytokines [Nestorova I.V., 1999; Karaulov A.B., 1999; Nestorova I.V., Starchenko A.A., Ivanova S.A., Simbirtsev A.S., 2002; Starchenko A.A., 2002].

At the same time, the choice of a preparation for targeted correction of this or that shift in the immune system in response to non-infectious extreme impact remains a sufficiently complex task, since in most cases attempts to isolate any "main defect" of the immune response in somatic patients are not feasible [Kozlov V.A., 1997; Karaulov A.B., 1999].

It should be noted that immunorehabilitation, as a direction of clinical immunology, is oriented toward the use of safe, accessible, commensurate with the patient's adaptive capabilities medicinal, physiotherapeutic, and sanatorium-resort methods of restoration of immune system functions [Shanin S.N., Rybakina E.G., Fomiceva E.E., 1999; Pershin B.B., Kuzmin S.N., Medvedev V.Ya., Kholstov D.V., 1999; Sepiashvili R.I., 1999; Yarenenko K.V., Ivanova S.A., 2002]. Therefore, alongside the elucidation of the mechanism of immunosuppressive states, the search for modifiers of biological reactions whose action is directed not only at immune but also other cells and systems of the organism is of practical significance.

These principles of immunorehabilitation are fully met by methods of phytotherapy. The use of medicinal plants appears to be a promising direction for the further development and improvement of methods of immunorehabilitation, since modern pharmacology does not possess a sufficient arsenal of effective means for the prevention, treatment of chronic diseases, stress, and its consequences [Shanin S.N., Rybakina E.G., Fomiceva E.E., 1999; Shanin S.N., Kozinets I.A., Fomiceva E.E., 1996].

Nevertheless, despite the widespread use of phytopreparations in folk medicine, the growing interest of physicians and researchers in studying the effects of natural compounds, the mechanisms of their influence on protective reactions of the organism (in particular, the immune and hematopoietic systems) under impacts of various natures, including stress, remain insufficiently studied, as evidenced by the scanty literature data [Shanin S.N., Rybakina E.G., Fomiceva E.E., 1999; Shanin S.N., Kozinets I.A., Fomiceva E.E., 1996].

According to modern concepts, extreme factors of the external environment are divided into two main groups: physico-chemical and information-semantic. A model of a typical physico-chemical extreme factor may be the toxic action of preparations used for the treatment of malignant neoplasms [Masnaya N.V., 1998; Goldberg E.D., Dygai A.M., Zhdanov V.V., 1999; Goldberg E.D., Dygai A.M., Sherstoboev E.Yu., 2000].

The most convenient, but, at the same time, insufficiently studied model for investigating the nature of dysfunction of the immune and hematopoietic systems under the influence of information-semantic extreme factors is experimental stress, which allows regulation of the direction and depth of changes in the functioning of these systems, as well as evaluation of the effectiveness of preparations claiming the role of correctors and modulators of their function. The use of this model for studying the peculiarities of functioning of the immune and hematopoietic systems of animals with different genotypes, differently reacting both to an antigenic stimulus and to stressor impacts, will make it possible to study the mechanisms of influence of extreme situations on the immune response, and the involvement of adequate pharmacological methods will allow effective search for methods of targeted correction of the detected violations. A convenient model of genetically determined differences in reaction to antigenic and stressor stimuli are inbred strain mice. Analysis of interstrain differences will make it possible to draw conclusions about the presence or absence of differences in the sensitivity of the hematopoietic and immune systems to extraordinary factors.

Goal of the work.

To investigate the peculiarities of impairments of the response of the immune and hematopoietic systems to a thymus-dependent antigen after immobilization stress in animals with different genotypes and to conduct correction of the detected violations using natural origin preparations.

Tasks of the research.

1. To investigate general patterns and peculiarities of functioning of the hematopoietic and immune systems of animals of different lines during immunization with a thymus-dependent antigen.

2. To study the peculiarities of reactions of the hematopoietic and immune systems of animals with different genotypes in response to stressor impact at various investigation time points.

3. To identify differences in the formation of the immune response of animals of different lines under immobilization stress.

4. To study the mechanisms of immunosuppressive states in animals of different lines under extreme impact.

5. To conduct correction of the immunodeficient state caused by immobilization stress in mice of the CBA/CaLac and DBA/2 lines using natural origin preparations.

Positions to be defended.

1. Administration of a thymus-dependent antigen leads to an increase in the absolute number of antibody-forming cells and the titer of specific antibodies in the initial observation periods after immunization in high-responding mice CBA/CaLac, DBA/2, BALB/c, and to a delay in the accumulation of antibody-producing cells in low-responding mice C57BL/6, CC57W. The specific humoral response after immobilization stress conducted against the background of immunization was depressed to a greater extent in high-responding (CBA/CaLac, DBA/2, BALB/c) than in low-responding (C57BL/6, CC57W) lines.

2. Immobilization stress changes not only the ratio of immunocompetent cells but also their functional activity (cytokine production, proliferative activity).

3. Administration of a thymus-dependent antigen leads to hyperplasia of hematopoietic and lymphoid organs of mice of the CBA/CaLac, DBA/2, BALB/c, C57BL/6, and CC57W lines.

4. In the bone marrow of immobilized mice CBA/CaLac, BALB/c, C57BL/6, and CC57W, activation of the lymphoid hematopoietic lineage is observed, erythroid — in CBA/CaLac and C57BL/6, myeloid — in BALB/c, C57BL/6, and CC57W. In the spleen, an increase in the number of the lymphoid hematopoietic lineage occurs in CC57W, CBA/CaLac, depression — in BALB/c, decrease with subsequent stimulation of the investigated lineage — in DBA/2 and C57BL/6. The erythroid lineage is stimulated by stress in all investigated mouse lines. The number of myeloid cells increases in CC57W and C57BL/6, decreases in BALB/c. In mice CBA/CaLac, DBA/2, and CC57W, involution of the thymus occurs, in BALB/c and C57BL/6 — increase in cellularity of this lymphoid organ.

5. In immobilized and immunized mice CBA/CaLac, DBA/2, and C57BL/6, depression occurs, in BALB/c and CC57W — activation of the lymphoid lineage in the bone marrow, decrease in the number of rosette-forming cells in C57BL/6 and increase — in BALB/c. The number of myeloid cells in the bone marrow increases in CBA/CaLac, BALB/c, and CC57W, and decreases — in C57BL/6. In the spleen, an increase in the number of the lymphoid lineage occurs in mice BALB/c, CC57W, and CBA/CaLac (after a brief decrease), a decrease — in C57BL/6. The myeloid lineage of the spleen is stimulated in BALB/c, C57BL/6, and CC57W. In mice CBA/CaLac, DBA/2, C57BL/6, and CC57W that underwent immunization and immobilization, involution of the thymus develops.

6. Administration of Altai hellebore extract to mice immunized against the background of transferred immobilization stress leads to depression of bone marrow and stimulation of splenic hematopoiesis, hyperplasia of the thymus. Use of pantocrin in mice immunized after transferred immobilization impact activates erythroid, myeloid, and lymphoid hematopoietic lineages.

7. Use of pantocrin, extracts of Altai hellebore, thick-leaved rhodiola rosea, ivy-leaved birdfoot trefoil, and creeping peony tincture after immobilization stress but before immunization contributes to restoration of the content of antibody-forming cells. Use of pantocrin, extract of Altai hellebore, and purple coneflower tincture stimulates the proliferative activity of splenic B-lymphocytes.

Scientific novelty.

For the first time, differences in the reaction of hematopoietic and lymphoid tissue to a thymus-dependent antigen in mice of different lines (CBA/CaLac, DBA/2, BALB/c, C57BL/6, CC57W) were identified and characterized. Pronounced differences were demonstrated in the formation of the humoral immune response in high-responding (CBA/CaLac, DBA/2, BALB/c) and low-responding (C57BL/6, CC57W) antigen lines of animals, which is expressed in a significant increase in the absolute number of antibody-forming cells in the spleen and the titer of antibodies in the blood serum in the initial periods after immunization of mice of the CBA/CaLac, DBA/2, BALB/c lines and a delay in the accumulation of antibody-producing cells after immunization of mice C57BL/6, CC57W. The established differences in the reaction of the immune system of mice to the antigen are based on the different functional activity of components of the immune response (macrophages, T- and B-lymphocytes, antibody-producing cells).

For the first time, it was demonstrated that immobilization stress depresses the development of the specific humoral response and phagocytic activity of peritoneal macrophages in immunized high-responding mouse lines (CBA/CaLac, DBA/2, BALB/c) and, exerting insignificant influence on the productive phase of the humoral immune response, depresses the activity of peritoneal macrophages in the group of low-responding mice (C57BL/6, CC57W). At the same time, a decrease in the number of total T- and rosette-forming B-lymphoid cells and an increase in the number of T-helpers in the bone marrow and spleen occurs in mice DBA/2 and C57BL/6.

A decrease in the number of S044-lymphocytes was revealed in all lines subjected to immobilization (except CBA/CaLac), and an increase in the number of SВ8+ cells in all lines except DBA/2. For the first time, it was established that under the influence of immobilization stress in mice CBA/CaLac, BALB/c, C57BL/6, a decrease in the proliferative activity of T- and B-lymphocytes in the spleen occurs, in mice DBA/2 — an increase in the stimulation index of ConA-stimulated T-lymphocytes. It was shown that against the background of development of the immune response, stress increases the level of IL-2 and decreases the synthesis of IL-10 in mice CBA/CaLac, in C57BL/6 there is an enhancement of synthesis of IL-2 and IL-10. It was established that synthesis of IL-2 and IL-10 is depressed in groups of mice CBA/CaLac and C57BL/6 that underwent immobilization stress, alongside a decrease in the ability of T-lymphocytes to produce proinflammatory cytokines in CBA/CaLac and a disruption of the processes of activation of antigen-specific lymphocytes and synthesis by them of cytokines in C57BL/6.

It was shown that immunization leads to hyperplasia of hematopoietic and lymphoid organs CBA/CaLac, DBA/2, BALB/c, C57BL/6, and CC57W. Immobilization stress activates bone marrow hematopoiesis at the expense of the lymphoid lineage in mice of the CBA/CaLac, BALB/c, C57BL/6, and CC57W lines, erythroid — CBA/CaLac and C57BL/6, myeloid — in BALB/c, C57BL/6, and CC57W. In the spleen, an increase in the number of the lymphoid hematopoietic lineage is observed in CC57W, CBA/CaLac, the erythroid lineage — in all investigated mouse lines. In mice CBA/CaLac, DBA/2, and CC57W, development of thymus hypoplasia is observed, in BALB/c and C57BL/6 — an increase in the number of lymphocytes of all degrees of maturity in the central lymphoid organ. For the first time, it was demonstrated that immobilization stress conducted against the background of a developing immune response depresses the lymphoid lineage in mice CBA/CaLac, DBA/2, and C57BL/6, and stimulates it in BALB/c and CC57W, alongside the myeloid lineage in BALB/c, CC57W, and CBA/CaLac. In the spleen, an increase in the number of lymphoid cells is observed in mice DBA/2, CC57W, and CBA/CaLac (after a brief decrease), a decrease — in C57BL/6. The myeloid lineage of the spleen is activated in BALB/c, C57BL/6, and CC57W. In mice CBA/CaLac, DBA/2, C57BL/6, and CC57W, development of thymus hypoplasia is observed.

For the first time, it was established that use of Altai hellebore extract and pantocrin restores hematopoiesis and lymphopoiesis depressed by immobilization impact. Use of a plant preparation in immunized mice against the background of transferred immobilization stress leads to development of depression of hematopoiesis and hyperplasia of the thymus. In the group of mice immunized after transferred immobilization impact, use of pantocrin activates the myeloid, lymphoid, and erythroid hematopoietic lineages in the bone marrow and spleen.

Use of pantocrin, extracts of Altai hellebore, thick-leaved rhodiola rosea, ivy-leaved birdfoot trefoil, and creeping peony tincture for correction of the immunodeficient state (after immobilization stress but before immunization) contributes to restoration of the content of antibody-forming cells and increase in their production of specific antibodies. Extracts of rhodiola rosea, ivy-leaved birdfoot trefoil, and purple coneflower tincture possess anti-inflammatory properties, reducing the inflammation index in the hypersensitivity reaction. Use of pantocrin, extract of Altai hellebore, and purple coneflower tincture stimulates the proliferative activity of LPS-stimulated B-lymphocytes in the spleen of experimental animals.

Practical significance of the work.

The results of the work significantly expand the representation of the peculiarities of reaction of the hematopoietic and immune systems to administration of a thymus-dependent antigen and immobilization stress in mice of the CBA/CaLac, C57BL/6, DBA/2, BALB/c, and CC57W lines. Differences in the reaction of the bone marrow, thymus, and spleen, as well as cells participating in the formation of the immune response in mice of different lines after immobilization stress have been demonstrated. Thanks to the obtained data, a contribution was made to understanding the general patterns and peculiarities of development of the immune response stimulated by a thymus-dependent antigen after immobilization stress. The principled possibility and pathogenetic justification of the use of natural origin preparations in the therapy of immunodeficient states have been demonstrated.

Presentation of the work.

The materials of the dissertation were reported and discussed at the VII All-Russian Symposium "Correction of Homeostasis" (Krasnoyarsk, 1996); at the conference "Actual Problems of Pharmacology and Search for New Pharmaceutical Preparations" (Tomsk, 1997); at the 3rd Congress of Physiologists of Siberia and the Far East (Novosibirsk, 1997); at the conference dedicated to the 15th anniversary of the Research Institute of Pharmacology of the Tomsk Scientific Center of the Siberian Branch of RAMN "Actual Problems of Pharmacology and Search for New Pharmaceutical Preparations" (Tomsk, 1999); at the international scientific conference "Search, Development and Implementation of New Pharmaceutical Means and Organizational Forms of Pharmaceutical Activity" (Tomsk, 2000); at the 2nd Russian Conference of Young Scientists of Russia with international participation "Fundamental Sciences and Progress of Clinical Medicine" (Moscow, 2001); at the scientific youth conference of the Siberian Branch of RAMN "Fundamental and Applied Problems of Modern Medicine" (Novosibirsk, 2001); at the conference "Actual Problems of Experimental and Clinical Pharmacology" (Tomsk, 2001, 2002); at the IV Congress of Physiologists of Siberia with international participation (Novosibirsk, 2002); at the IV youth scientific conference of the Siberian Branch of RAMN "Fundamental and Applied Problems of Modern Medicine" (Novosibirsk, 2002); at the All-Russian conference "Compensatory-Adaptive Processes: Fundamental and Clinical Aspects" (Novosibirsk, 2002); at the conference "Actual Problems of Pharmacology" (Tomsk, 2004).

Publications.

42 scientific works have been published on the topic of the dissertation, of which 13 are in central refereed journals.

Volume and structure of the work.

The dissertation is presented in two volumes. Volume I is 390 pages of typed text and consists of the introduction, four chapters, conclusions, and the list of references. Volume II (appendix) is 122 pages. The work is illustrated by 47 figures and 124 tables (tables 15-124 are placed in the appendix). The bibliographic index includes 510 sources, of which 379 are domestic and 131 are foreign.