Sergei V. Khaidukov

Comparative Analysis of T Lymphocytes Recovered from the Lungs of Mice Genetically Susceptible, Resistant, and Hyperresistant to Mycobacterium tuberculosis-Triggered Disease

Genetic control of susceptibility to tuberculosis (TB) is being intensively studied, and immune responses to mycobacteria are considerably well characterized. However, it remains largely unknown which parameters of response distinguish resistant and susceptible TB phenotypes. Mice of I/St and A/Sn inbred strains and (A/Sn × I/St)F1 hybrids were previously categorized as, respectively, susceptible, resistant, and hyperresistant to Mycobacterium tuberculosis-triggered disease. In the present work we compared parameters of lung T cell activation and response following M. tuberculosis challenge. In all mice, the disease progression was accompanied by a marked accumulation in the lungs of activated CD4+ (CD44high/CD45RBlow) and CD8+ (CD44high/CD45RB+) T cells capable of secreting IFN-γ and of activating macrophages for NO production and mycobacterial growth inhibition. However, significantly more CD8+ T cells were accumulated in the lungs of resistant A/Sn and F1 compared with I/St mice. About 80% A/Sn and F1 CD8+ cells expressed CD44high/CD45RB+ phenotype, while about 40% I/St CD8+ cells did not express CD45RB marker at week 5 of infection. In contrast, in susceptible I/St mice lung CD4+ cells proliferated much more strongly in response to mycobacterial sonicate, and a higher proportion of these cells expressed CD95 and underwent apoptosis compared with A/Sn cells. Unseparated lung cells and T cells of I/St origin produced more IL-5 and IL-10, respectively, whereas their A/Sn and F1 counterparts produced more IFN-γ following infection. F1 cells overall expressed an intermediate phenotype between the two parental strains. Such a more balanced type of immune reactivity could be linked to a better TB defense.

Intranasal BCG vaccination protects BALB/c mice against virulent Mycobacterium bovis and accelerates production of IFN-γ in their lungs

Local immune reactivity in the lungs of BALB/c mice was studied following (i) intranasal (i.n.) vaccination with Mycobacterium bovis BCG, (ii) intravenous (i.v.) challenge with a virulent M. bovis field isolate and (iii) i.n. vaccination with M. bovis BCG followed by i.v. challenge with an M. bovis field isolate. The results demonstrated that i.n. vaccination with BCG induced a high degree of protection against systemic M. bovis challenge, and that this protection correlated with a rapid production of IFN‐γ after M. bovis challenge by lung T cells from vaccinated mice.

Anti-DNA autoantibodies reveal toxicity to tumor cell lines.

Cytotoxicity of anti-DNA autoantibodies from sera of SLE and CLL patients was assayed on permanent cell lines L929, HL-60, Raji, and K562. L929 cells appeared to be the most sensitive to antibody treatment. DNA-hydrolyzing properties of the same autoantibody preparations were analyzed in parallel. The data obtained outlined the correlation between cytotoxicity and DNA-hydrolyzing properties of these autoantibodies. It was shown that treatment of the cells with cytotoxic anti-DNA autoantibodies induced internucleosomal DNA fragmentation and Annexin V binding to the cell surface characteristic of apoptotic pathway of cell death. A time-dependent profile of antibody-mediated toxicity to L929 cells suggested recruitment of at least two distinct mechanisms of cell death. The first peak of cell death observed in 3 h of incubation was completely inhibited by preincubation of cells with caspase inhibitor YVAD-CHO, while the second increase in cell mortality (18-30 h) persisted. Possible mechanisms for anti-DNA autoantibody cytotoxicity are discussed.

Time-dependent changes of LAK cell phenotypes correlate with the secretion of different cytotoxic proteins.

Human lymphokine-activated killer (LAK) cells were generated from peripheral blood lymphocytes (PBL) of normal volunteers by interleukin-2 (IL-2) stimulation for 1-8 days. During the first 3 days the surface marker CD16 characteristic for natural killer (NK) cells was expressed and later the CD3 marker characteristic for cytotoxic T cells became predominant. The conditioned media of LAK cells collected after interaction of LAK cells with K562 target cells was chromatographically separated into two cytotoxic fractions: F1 and F2. It was demonstrated that fraction F1 contained cytotoxic proteins having molecular weights of 30 and 40 kDa, and fraction F2 contained cytotoxic proteins having molecular weights of 22, 38 and 75 kDa. The presence of the proteins in each of these two fractions correlated with the phenotype changes of LAK cells: the F2 cytotoxic proteins were characteristic for NK-like cells, and the F1 proteins for cytotoxic T-lymphocyte (CTL)-like phenotypes.

Characterization of T cell clones derived from lymph nodes and lungs of Pseudomonas aeruginosa-susceptible and resistant mice following immunization with heat-killed bacteria

Pseudomonas aeruginosa‐resistant BALB/c and susceptible C57Bl/6 (B6) mice were immunized with heat‐killed Pseudomonas either in the foot pad or via the trachea, and panels of Pseudomonas‐specific T cell clones were developed from lymph nodes and lungs. All clones from either strain, whether of lymph node or lung origin, were CD3+CD4+CD8−TCRαβ+. The efficacy of cloning from lymph node cells was comparable between BALB/c and B6 mice. All lymph node BALB/c clones proliferated in response to Pseudomonas antigen in a dose‐dependent manner, and this response was MHC class II‐restricted. Vigorous proliferation by a considerable proportion of B6 T cell clones occurred in the absence of specific antigen. Lymph node clones from either strain could be categorized as either Th1 or Th0 on the basis of interferon‐gamma (IFN‐γ)/IL‐4 production. In either mouse strain the efficacy of cloning from lung tissue was substantially lower than from lymph nodes, but the efficacy of cloning from BALB/c compared with B6 lungs was higher. Four lung T cell clones from BALB/c and two from B6 mice were expanded for further analyses, and an interstrain difference was observed in cytokine production. Both B6 lung T cell clones were Th1‐like and produced IFN‐γ but not IL‐4 and IL‐10, whereas four BALB/c lung T cell clones were Th2‐like and produced IL‐4 and IL‐10 but not IFN‐γ. These observations suggest that differences in the CD4+ Th response in the lung may contribute to differences among inbred mouse strains in the level of resistance to bronchopulmonary Pseudomonas infection.

Capacity of murine T cells to retain long-term responsiveness to mycobacterial antigens is controlled by the H-2 complex

It is firmly established that the allelic composition of the H‐2 complex has a prominent impact on the course of tuberculosis (TB) infection in mice, including granuloma formation, mycobacterial spread in the lungs, and the dynamics of mortality. Although intuitively obvious, the role of long‐term specific T cell responses in the expression of corresponding phenotypes is poorly understood. In this study we have compared polyclonal lymph node cell response (cell yield, proliferation, surface markers, IL‐4/interferon‐gamma (IFN‐γ) production) to Mycobacterium tuberculosis H37Rv sonicate in repeated 10‐day cycles of stimulation/rest between H‐2 congenic IE‐negative mouse strains, categorized on the basis of mortality following lethal challenge as TB‐susceptible (C57Bl/6), TB‐resistant (4R) and BCG non‐protected (B10.M). The capacity to retain specific responsiveness to repeated stimulation by mycobacterial antigens depended upon both the H‐2 haplotype of the host and the immunizing dose of the antigen. 4R lymph node cells following either 50 μg/mouse or 100 μg/mouse immunization constantly responded to sonicate, increased in numbers, and after the third stimulation/rest cycle developed into a stable CD3+ CD4+ cell line. B6 cells following either 50 μg/mouse or 100 μg/mouse immunization, and B10.M cells following 100 μg/mouse (but not 50 μg/mouse) immunization, lost the capacity to incorporate methyl‐3H‐thymidine during the second cycle, and died. Analogous results were obtained in the in vivo experiments, when the dynamics of the response over 12 weeks following a single immunization with the antigen was studied. In response to the antigen, cells from all three mouse strains produced significant amounts of IL‐2 and IFN‐γ, but not IL‐4, indicating that they belong predominantly to the Th1‐like subset. Among noteworthy differences between the mouse strains was a clear deficiency of CD8+ T cells in B6 cultures, and an unusually high proportion of CD3+ CD4− CD8− (double‐negative) T cells in B10.M cultures following a high‐dose immunization.

Endogenous fragment of hemoglobin, neokyotorphin, as cell growth factor.

It is shown that neokyotorphin (the alpha-globin fragment 137-141) stimulates proliferation of normal cells (murine embryonic fibroblasts, red bone marrow and spleen cells) and tumor cells (murine melanoma and transformed fibroblasts L929) in the absence or in the presence of fetal bovine serum. In contrast to serum deprivation conditions, the ability to potentiate L929 cell growth in the presence of fetal serum is strongly cell density dependent. The peptide also enhances the viability of L929 cells, murine embryonic fibroblasts and of the primary cultures of murine red bone marrow cells and splenocytes under serum-deprivation conditions for at least 72 h. The results of flow cytometry analysis suggest that the effect of neokyotorphin on survival of L929 cells in serum-free culture medium is due to maintenance of cell proliferation in the absence of growth factors. Along with cell cycle progression the peptide induces reversible reduction of L929 cell size.

Caspase-Dependent Cytotoxicity of Anti-DNA Autoantibodies

The role of the catalytic function of natural antibodies in the organism is still unclear. It is commonly accepted that occurrence of natural antibodies is associated with the development of human autoimmune diseases (e.g., lupus erythematosus; LE) and autoimmune conditions in model mouse strains NZBxNZW and MRL/lpr. First of all, this dependence is characteristic of DNA-hydrolyzing antibodies. We discovered that the DNA-hydrolyzing antibodies are toxic for transplantable cell lines and analyzed pathway of cell death caused by these antibodies.

The Modification of Cell Surface with Lipophilic Glycoconjugates and the Interaction of Modified Cells with Natural Killer Cells

An experimental model system involving the modification of carbohydrate composition of the target cell surface with neoglycolipids was developed for studying the role of surface carbohydrates of target cells in the NK-cell-mediated cytotoxicity. The polymeric glycoconjugates of the Glyc–PAA–PEA and Glyc–PAA(Flu)–PEA types (where Glyc was an oligosaccharide residue, PAA poly(acrylamide) polymer, PEA the phosphatidylethanolamine residue, and Flu fluorescein residue) capable of incorporating into the cell membrane were synthesized. The optimum structures of neoglycoconjugates and the conditions for their incorporation into K562 and Raji cell lines, which differ in their sensitivity to the NK-cell-mediated lysis were selected. The mechanism of association of glycoconjugates with the plasma cell membrane and the kinetics of their elimination from the cell surface were investigated using the fluorescent-labeled Glyc–PAA(Flu)–PEA derivatives. The spatial accessibility of the carbohydrate ligands for the interaction with human NK cells was demonstrated. The target cells modified with the Lex trisaccharide were shown to be more sensitive to the cytotoxic effect of human NK cells than the intact cells.

In vivo Proteolytic Hemoglobin Products as Tissue Growth Promoters

It is well known that growth factors are responsible for maintenance of cell proliferation in vitro and in vivo. At the same time, the ability to stimulate cell proliferation in vitro was demonstrated for endogenous fragments of functional proteins, defined earlier as components of tissue-specific peptide pools [1]. Neokyotorphin, the a-globin (137–141) fragment, has been shown earlier to stimulate proliferation of L929 transformed murine fibroblasts and brown preadipocytes independently on the presence of fetal bovine serum (FBS) [2–4]. We have studied the in vitro proliferative effects of the peptides corresponding to the a-globin (133–141) segment, and on the basis of the data obtained suggested their function in the organism.