LABORATORY OF CELL INTERACTIONS
Division of Immunology
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry
A.M. Sapozhnikov, Ph.D.
Investigators: Dr. G.V. Lutsenko, Dr. I.M. Molotkovskaya, Dr. E.V. Svirshchevskaya, Dr. E.I. Kovalenko, Dr. N.I. Lutsan, L.G. D’yachkova, N.Yu. Viskova, N.A. Zelenova, I.G. Pushilina, E.G. Frolova, E.D. Ponomarev
The main fields of the laboratory are the following: (i) the control mechanisms of immunocompetent cell activation; (ii) peptide-induced antigen-specific anergy of T lymphocytes; (iii) the role of autocrine/paracrine factors in cell stress and programmed cell death; (iv) assay of immunomodulators.
Regulation of immunocompetent cell activation
Gangliosides have been shown to inhibit proliferation of interleukin-2 (IL-2)-dependent cell line CTLL-2. As we have found in collaboration with the Laboratory of Lipid Chemistry previously, more than one type of cell growth inhibition is realized by six different gangliosides. To continue this work we have studied the influence of different glycosphingolipids (GM1, GD1a, GT1b, GM3, GD3 and lactosylceramide) on proliferation of IL-4-dependent cell line CT.4R. The purpose of this investigation was to differentiate the effects of various gangliosides on cell growth and to characterize their interaction with IL-4 and IL-4 receptor . Using the fluorescence-labelled gangliosides as probes we have demonstrated the specific binding of the IL-4 molecule to gangliosides in a model membrane system. With help of this model we have also defined the dissociation constants for the complexes of GM1, GM3 and GD1a with IL-4.
The role of serum factors in mitogen-induced proliferation of resting T cells was studied. It was carried out the investigation of the influence of fetal calf serum on concanavalin A (ConA)-stimulated murine T lymphocyte progression through the initial and following cell cycles. We have found that resting T lymphocytes treated by Con A are able to progress through the initial cycle in the absence of serum factors. Moreover, the sensitivity of cells to ConA was decreased cycle in the presence of fetal serum, doing this effect more pronounced with increasing its concentration. On the contrary, the progression of the cells through the second and next cycles was possible only in the medium supplemented with more than 10% of serum. It is important that the ability of T lymphocytes for progression through the initial cell cycle without serum correlates with known ability of activated T cells for the initial progression without IL-2. These facts suggest that the mechanism of activation of resting T cells differs from the mechanism of following support of their proliferation.
Investigation of the role of autocrine/paracrine factors produced by activated T lymphocytes in regulation of their metabolism was continued. It was revealed that T lymphocytes from exponentially growing cultures with high density adapted during one cell cycle to increased level of autocrine/paracrine factors controlling energy metabolism. The transfer of these cells to low density cultures resulted in their fast death by apoptosis mechanism. Increased concentrations of serum or autocrine factors (obtained by using the conditioned supernatant) were needed for their survival. Adaptation of T cells to increased levels of these factors was reversible, since the requirement of cells for the factors could be decreased for several hours. The data were obtained suggesting that both serum and autocrine factors controlled metabolism of glucose.
The role of cell membrane chloride channels in energy metabolism and proliferation of lymphoid tissue was studied. We found that effect of chloride channel blockers 4-acetamido-4-isothiocyano-2,2’-disulfonic acid stilbene (SITS) and 4,4’-diisothiocyano-2,2’-disulfonic acid (DIDS) was dependent on the level of cell maturation. Our results showed that SITS and DIDS inhibited energy metabolism of mature ConA-activated murine splenocytes but significantly stimulated that of bone marrow haemopoietic precursors. At the same time we revealed the dependence of cell response to SITS and DIDS on cell culture density. This indicates that autocrine/paracrine factors can affect the action of Cl- -channel blockers and it may suggests that different production of such factors by activated T lymphocytes and by bone marrow cells resulted in their different reaction to decrease of cell membrane permeability for chloride ions.
Identification of immunodominant peptide fragments of allergens
from Aspergillus fumigatus
We have carried out the screening of five synthetic oligo peptides - hypothetical immunodominant epitopes of some major allergens of Aspergillus fumigatus (Af), a ubiquitous fungus implicated in the pathogenesis of a number of allergic diseases (including allergic asthma) in man (joint investigation with the Laboratory of Peptide Chemistry). The goal of this investigation is the development a new approaches to immunotherapy of allergy, based on the use of T cell epitope-containing peptides inducing anergy of antigen-specific T lymphocytes. It was demonstrated that among five peptides, only one (Af-1 128-140) induced pronounced proliferative response of Af-specific murine T lymphocytes and could support in vitro production of Af-specific antibodies by immune splenocytes. This indicates that this peptide contain T epitope of Af. Another peptide (Af-1 87-99) could not induce proliferation of immune T cells but bound antibodies from immune serum with highest titer, that allowed to consider this fragment as B epitope-containing. These results will be used for study of peptide-induced T cell anergy in vitro.
The role of autocrine/paracrine factors in cell stress
and programmed cell death
The study of influence of cell produced autocrine/paracrine factors on the stress-induced proteins (heat shock proteins - HSPs) expression and programmed cell death (apoptosis) was performed. In the investigation we used the model of spontaneous apoptosis in vitro of mouse lymphoma cells EL-4 expressed surface HSPs. We registered the level of surface HSPs and percentage of apoptotic cells in cultures with different density (with different number of cultivated cells). It was revealed the increase of percentage of apoptotic cells in cultures with low and high density. The level of surface HSPs was greatest in cultures with reduced concentration of cytokines (in low density cultures) that allowed us to consider lack of autocrine/paracrine factors as stress for cells. This was confirmed by fact that low density cultures supplemented with enriched by such cytokines conditioned supernatant displayed the percentage of apoptotic cells and level of surface HSPs typical of cells in conventional conditions. At the same time, conditioned supernatant acted in a similar manner on cell energy metabolism in low density cultures. This results suggest involvement of factors affecting cell stress and apoptosis in control of cell energy metabolism.
Assay of immunomodulators
The study of immunomodulatory properties of GMDP and synthetic myelopeptides MP1, MP2, MP3 and MP4 was continued.
Synergetic action of GMDP and thymic hormones on the activation and maturation of thymocytes and bone marrow precursors of T cells was demonstrated (joint investigation with the Laboratory of Peptide Chemistry). This confirms the supposition about possibility of the use of conjugates and compositions of GMDP with cell-specific ligands for selective stimulation of certain type of cells. Besides, the investigation of influence GMDP on early stages of cell activation was carried out. It was found that preincubation of murine B cells with GMDP resulted in significant increase of Ca2+ influx in these cells after activation by anti-mouse IgG antibodies. These results are in conformity with the effect of GMDP on mitogen-induced cell proliferation.
The mechanisms of stimulating action of MP1 on mitogen-induced activation of T cells of old mice was studied (joint investigation with the Laboratory of Immune System Mediators). Our data showed that sensitivity of Ca2+ transport system of T lymphocytes of old mice to ConA increased after the treatment of cells by MP1 as much as the level of that of young mouse cells. For another myelopeptide - MP2 it was demonstrated its ability to substitute serum factors regulated T cell proliferation. At the same time we displayed that MP2, MP3 and MP4 could compensate lack of autocrine/paracrine factors in low density cultures of mouse lymphoma EL-4. The investigation of the action of myelopeptides on lymphocyte differentiation revealed that MP1 and MP3 stimulated the maturation of B cells in bone morrow cell culture. It was evaluated also the effect of MP3 on production of some cytokines by macrophages. In these experiments we showed the simulating action of this peptide on secretion of TGF-betta and absence of influence of MP3 on production of TNF-alfa.
References
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