Method to evaluate the proliferation of activated lymphocytes in a three-dimensional collagen matrix.

It is well known that the enhancement of the cell-matrix interactions represents one of the early steps in the process of lymphocyte activation. However, the information regarding the role of these interactions in the late stages of lymphocyte activation (in particular, the proliferation) is still controversial. This is basically due to the absence of adequate experimental models. In the present work we carried out a step-by-step modification of a well-studied model of mitogen-stimulated lymphocyte activation, adjusting it to the conditions of a three-dimensional collagen matrix (3D-CM). All the changes added to the standard procedure in the process of this modification were rigorously controlled using various experimental models. The final version of the method includes the following steps: (i) 24-h lymphocyte (lymphocyte fraction from mouse spleen) preincubation with mitogens (Con A or LPS) with a subsequent cell wash (parameters being controlled: irreversible lymphocyte activation, independence of the proliferation from cell-cell interactions); (ii) transfer of the activated lymphocytes to (3)H-thymidine containing 3D-CM and incubation for 48 h (controlled parameters: distribution of the radioactive label within the 3D-CM and its biological accessibility to lymphocytes); (iii) degradation of the 3D-CM with bacterial collagenase and cell transfer onto glass fiber filters (controlled parameters: cell viability after cultivation in the 3D-CM and treatment with the collagenase). With this method we found that the proliferation of the Con A- and LPS-stimulated lymphocytes in 3D-CM was dramatically inhibited (by 66.5 +/- 14.9% and by 88.1 +/- 10.2%, respectively). The discovered inhibition of the lymphocyte proliferation was not a consequence of either the ineffectiveness of the mitogens, the disruption of the cell-cell interactions, an insufficient inclusion of the radioactive label into cells, or of a decreased cell viability.

Binding of alpha2-macroglobulin to collagen type I: modification of collagen matrix by alpha2-macroglobulin induces the enhancement of macrophage migration.

Alpha2-macroglobulin (a2M) secreted by tissue macrophages and fibroblasts functions in the environment of extracellular matrix macromolecules. We supposed that it may interact with these molecules and change the properties of extracellular matrix. Modified variant of ELISA was used to prove the direct binding of human a2M to collagen. Native and transformed by plasmin a2M, as well as plasmin, used as the control, were labeled by biotin. It has been found that the transformed, but not the native a2M form binds to type I collagen molecules: K(d)=(1.168 +/- 1.14) x 10(-11) M. The data obtained give a strong evidence of high power of the interaction between a2M and type I collagen: practically no reverse dissociation may be seen for such a binding. The modification of three-dimensional collagen matrix by binding to the transformed a2M resulted in the enhancement of migration of macrophages, carrying the receptors for a2M, but not splenocytes that lack for such receptors. Our results allow to suggest that a2M may be one of the components of extracellular matrix, and may change the properties of microenvironment for immunocompetent cells during the processes of inflammation, reparation and tumor invasion.

Alpha2-Macroglobulin Modulates Interactions between Lymphocytes and Fibroblasts.

The aim of the present study was to evaluate the influence of apha2-macroglobulin (alpha(2)M) on lymphocyte adhesion to fibroblasts. Peripheral blood lymphocytes from healthy donors and two fibroblast lines (human diploid embryo fibroblasts M-19 and mouse transformed fibroblasts L929) were used in the experiments. alpha(2)M treatment of fibroblast monolayer appeared to result in the enhancement of lymphocyte adhesion to fibroblasts. The number of attached lymphocytes was increased by 2-2.5 times. It should be noted that the effect of alpha(2)M didn't depend on the conformational molecule changes, since either native or methylamine or plasmin transformed alpha(2)M approximately at the same fashion increased the lymphocyte adhesion to both allogeneic and xenogeneic fibroblasts. B lymphocytes were predominant cells that were attached to fibroblast monolayer without alpha(2)M treatment. However the percentage of adherent T lymphocytes was increased substantially after the fibroblast monolayer treatment by alpha(2)M. Subpopulation analysis has shown that fibroblast pretreatment by alpha(2)M didn't result in a selective adhesion of CD4(+) or CD8(+) T lymphocytes, but increased the adhesiveness for both T lymphocyte subpopulations. The data obtained demonstrate that besides its participation in the processes of fibroblast adhesion alpha(2)M is capable to modify the contact interaction of these cells with lymphocytes that may have an influence on the functional consequences of this process.

Adhesion of Fibroblasts to Immobilized alpha(2)-Macroglobulin.

This study examines effects of alpha(2)-macroglobulin (alpha(2)M) on adhesion of fibroblasts. Native alpha(2)M and transformed form of alpha(2)M, alpha(2)M-plasmin, were bound to plastic. Adhesion of mouse L929 and human embryo M-19 fibroblasts to immobilized alpha(2)M was estimated under various conditions by counting adherent cells using videomicroscopy and computer-assisted image analysis. alpha(2)M-plasmin, bound to plastic, induced adhesion and spreading of mouse L929 and human M-19 fibroblasts. Neither native alpha(2)M nor plasmin alone did not induce fibroblast adhesion. The adhesion to alpha(2)M-plasmin was undetectable at 4 degrees C, as well as when sodium azide was added or divalent ions were removed. These findings provide novel information on alpha(2)M functions. On the basis of these observations we hypothesized that alpha(2)M, immobilized in the extracellular matrix, can participate in the regulation of microenvironment effects on the cells, and, in particular, influence on fibroblast adhesion.

Apoptogenesis of Immunodeficiency Diseases.

The tremendous progress, which has been made in the study of cellular and molecular basis of the maturation and functioning of immune system allowed to reveal the fine pathogenetic mechanisms of many primary (genetically caused) immunodeficiencies in human. The programmed cellular death is well known to be the basic natural mechanism of positive and negative selection in T and B lymphocytes, directed to elimination of cells with defective antigen-cognitive receptors or with capacity to react against "self". The controlled apoptosis is considered to be an important mechanism for support of optimal balance in the immune system. We designate immune deficiencies, in which the enhanced (pathological) apoptosis of cells of immune system take place, as apoptotic immunodefiencies. First of all, it means the apoptosis defects in T lymphocytes and their subpopulations. Triggering signals of apoptosis are highly variable. Among them it is possible to underline the specialized signals, acting through destined for them ligands. Moreover, the parameter of apoptosis may deserve as a criterion of severity of immunodeficiency process. The enhancement of spontaneous or induced apoptosis in lymphocytes, and first of all in T lymphocytes, may be considered as a marker of apoptotic immunodeficiency. The development of antiapoptotic immunocorrective drugs is the most important goal for clinical immunology. It is quite possible that the problem may be solved on the base of cytokines (growth factors), such as interleukin-2, granulocyte-macrophage colony-stimulating factor and others.

Low-Molecular Collagen Peptides Have Different Effects on Peritoneal Macrophages and Neutrophils.

Two types of phagocytes - neutrophils and macrophages, are very important participants in inflammation. However, the roles played by these cells in the regulation of an inflammation are radically different. Neutrophils initiate and ensure the alteration phase. Macrophages, to the contrary, regulate the transition of an inflammation from alternative processes to reparative. During the early stages of an inflammation, under the effect of proteases and free radicals, destruction of collagen proteins occurs and a large number of low-molecular peptides are formed, the concentration of which changes as the inflammatory reaction develops. The object of this work was to study the effect of the total fraction of low-molecular type I collagen peptides on the key functions of neutrophils and macrophages. Under the action of the wide range of concentrations of the collagen peptides (1-1000 &mgr;g/ml), activation of the neutrophil migration into the three-dimensional collagen matrix, amplification of PMA-induced production of free radicals and reduction of apoptosis of those cells were observed. The action of collagen peptides on the functions of macrophages had the opposite effect, i.e. they caused inhibition of the macrophage migration and reduction of PMA-induced production of free radicals. Furthermore, at a concentration of 100 &mgr;g/ml the collagen peptides reliably reduced the apoptosis of macrophages. Thus, collagen peptides are potent regulators of an inflammation, promoting the successive development of its phases through regulation of the functional state of phagocytes.

Low Molecular Weight Collagen Peptides Affect Migration, Proliferation and Apoptosis of Mouse Spleen Lymphocytes.

As a consequence of inflammatory tissue degradation collagen proteolysis products may be accumulated in the altered tissue. In this connection, we elaborated a hydrolysis scheme to obtain low molecular weight collagen peptides analogous to those produced in vtiro. To elucidate a possible role of collagen peptides during inflammation their action on lymphocyte migration, proliferation and apoptosis was studied at a wide range of concentrations 1-1000 &mgr;g/ml. The observed effects of peptides were different in three concentration ranges - low (1-50 &mgr;g/ml), middle (50-250 &mgr;g/ml) and high (250-1000 &mgr;g/ml). At high concentrations collagen peptides inhibited lymphocyte migration into 3D collagen matrix, and proliferation, including both spontaneous and stimulated. The middle peptide range induced lymphocyte apoptosis and modulated proliferation. Similar to middle ones, low concentration of collagen peptides modulated lymphocyte proliferation and their effect was the most pronounced. The three concentration ranges may presumably fit different stages of inflammation, since collagen degradation is associated with intensity of tissue alteration. Hence, collagen peptides may control lymphocyte functioning at different inflammation stages. Being naturally produced due to inflammatory tissue degradation, collagen peptides may be considered as complex inflammatory regulator like other traditionally discussed mediators (cytokines, chemokines, lipid mediators, etc.).

Pathogenetic Principle of Immune System Evaluation in Human: Positive and Negative Activation.

The main advantage of the pathogenetic principle for immune system evaluation is that the principle allows to optimize assessment of the main and accessory functions of immunocompetent cells. The investigation of immune system begins from the evaluation of cell recognition function, and then, step by step, the investigator moves further, while determining the capacity of immunocompetent cells to be activated, to proliferate and differentiate. The investigation of immune system may be terminated at its every stage once a defect in immune system is detected. Activation is one of the most important stage of cell life. Cell activation may be positive (complete functional cell cycle) or negative (apoptosis). It is suggested to pay attention to identification of defects in both positive, and negative chains of activation, since enhanced negative activation may be associated with different types of immunodeficiencies, whereas the absence of apoptosis (high positive activation) likely may be a cause of autoimmune and lymphoproliferative disorders.