N. V. Gnuchev

Novel functional activities of anti-DNA autoantibodies from sera of patients with lymphoproliferative and autoimmune diseases.

DNA-hydrolyzing activity of IgG autoantibodies from sera of patients with various types of lymphoproliferative diseases was investigated. The association of DNA-hydrolyzing activity with the antibody (Ab) fraction has been proved by newly developed affinity-capture assay. Study of abzyme incidence in blood tumors and systemic lupus erythematosis (SLE) revealed linkage of anti-DNA Ab catalysts to mature B-cell tumors, and increased probability of DNA-abzymes formation on the background of autoimmune manifestations. These data suggest possible similarity between mechanisms of abzyme formation in SLE and B-cell lymphomas. A new mechanism of formation of DNA-specific catalytic Abs has been proposed based on the increased crossreactivity of polyclonal DNA-abzymes to DNA-depleted nuclear matrix proteins. The possibility of the abzyme production as Ab to the energetically destabilized ground state of the antigen has been discussed. Preliminary results were obtained that indicate the complement-independent cytotoxicity of anti-DNA autoantibodies isolated from blood of patients with SLE and chronic lymphocytic leukemia.

Peptidoglycan Recognition Protein Tag7 Forms a Cytotoxic Complex with Heat Shock Protein 70 in Solution and in Lymphocytes

The peptidoglycan recognition protein Tag7 is shown to form a stable 1:1 complex with the major stress protein Hsp70. Neither protein is cytotoxic by itself, but their complex induces apoptotic death in several tumor-derived cell lines even at subnanomolar concentrations. The minimal part of Hsp70 needed to evoke cytotoxicity is residues 450–463 of its peptide-binding domain, but full cytotoxicity requires its ATPase activity; remarkably, Tag7 liberated from the complex at high ATP is not cytotoxic. The Tag7-Hsp70 complex is produced by tag7-transfected cells and by lymphokine-activated killers, being assembled within the cell and released into the medium through the Golgi apparatus by a mechanism different from the commonly known granule exocytosis. Thus, we demonstrate how a heat shock protein may perform functions clearly distinct from chaperoning or cell rescue and how peptidoglycan recognition proteins may be involved in innate immunity and anti-cancer defense.

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.

Opposite roles of metastasin (S100A4) in two potentially tumoricidal mechanisms involving human lymphocyte protein Tag7 and Hsp70

We compare the physical and functional interactions between three widespread multifunctional proteins [metastasin (Mts1/S100A4), innate immunity-related Tag7/PGRP-S, and Hsp70] in two experimental models relevant to host–tumor relationships on humoral and cellular levels. (i) Tag7 and Hsp70 in solution or in a lymphocyte make a stable binary complex that is highly cytotoxic for some tumor cells. Here, we show that Mts1 prevents Tag7·Hsp70 assembly in solution, and an excess of Mts1 disrupts the existing Tag7·Hsp70 complex; accordingly, Tag7·Hsp70 cytotoxicity (exemplified with L929 cells) is diminished in the presence of excess Mts1. (ii) Tag7 exposed on a specialized subset of lymphokine-activated killer cells makes specific contact with Hsp70 exposed on some HLA-negative tumor cells, thus enabling FasL/Fas-mediated induction of apoptosis. Here, we show that some CD4+CD25+ cells coexpose Mts1 with Tag7 and FasL, that Mts1 and Tag7 closely contact the same Hsp70 molecule on the target K562 cell (as evidenced by cross-linking), and that killing of such targets is abolished by Mts1-specific antibodies (or selective removal of Mts1-exposing lymphocytes). Thus, this phenotype active against immunoevasive cancerous cells is defined as CD4+CD25+, FasL+, Tag7+Mts1+ (≈0.5% of total lymphocytes in culture). Remarkably, similar effectors with at least the same activity are often found in fresh donor blood samples (≈104 effectors/mL). Thus, our models suggest that interactions between the three proteins in different situations may have opposite functional outcomes as regards antitumor defense, immune escape, and metastasis.

Tag7 (PGLYRP1) in Complex with Hsp70 Induces Alternative Cytotoxic Processes in Tumor Cells via TNFR1 Receptor.

Background: A complex containing an innate immunity protein Tag7, and Hsp70 kills various cancer cells. Results: Tag7 and its complex with Hsp70 bind to the TNFR1 receptor, but only the Tag7-Hsp70 complex induces a cytotoxic effect via apoptosis and necroptosis. Conclusion: A new ligand has been found for the TNFR1 death receptor. Significance: Tag7 may be used as an inhibitor of the TNF-α-induced cytotoxicity. Tag7 (also known as peptidoglycan recognition protein PGRP-S, PGLYRP1), an innate immunity protein, interacts with Hsp70 to form a stable Tag7-Hsp70 complex with cytotoxic activity against some tumor cell lines. In this study, we have analyzed the programmed cell death mechanisms that are induced when cells interact with the Tag7-Hsp70 complex, which was previously shown to be released by human lymphocytes and is cytotoxic to cancer cells. We show that this complex induces both apoptotic and necroptotic processes in the cells. Apoptosis follows the classic caspase-8 and caspase-3 activation pathway. Inhibition of apoptosis leads to a switch to the RIP1-dependent necroptosis. Both of these cytotoxic processes are initiated by the involvement of TNFR1, a receptor for TNF-α. Our results suggest that the Tag7-Hsp70 complex is a novel ligand for this receptor. One of its components, the innate immunity protein Tag7, can bind to the TNFR1 receptor, thereby inhibiting the cytotoxic actions of the Tag7-Hsp70 complex and TNF-α, an acquired immunity cytokine.

The heat shock binding protein (HspBP1) protects cells against the cytotoxic action of the Tag7-Hsp70 complex

Heat shock-binding protein HspBP1 is a member of the Hsp70 co-chaperone family. The interaction between HspBP1 and the ATPase domain of the major heat shock protein Hsp70 up-regulates nucleotide exchange and reduces the affinity between Hsp70 and the peptide in its peptide-binding site. Previously we have shown that Tag7 (also known as peptidoglycan recognition protein PGRP-S), an innate immunity protein, interacts with Hsp70 to form a stable Tag7-Hsp70 complex with cytotoxic activity against some tumor cell lines. This complex can be produced in cytotoxic lymphocytes and released during interaction with tumor cells. Here the effect of HspBP1 on the cytotoxic activity of the Tag7-Hsp70 complex was examined. HspBP1 could bind not only to Hsp70, but also to Tag7. This interaction eliminated the cytotoxic activity of Tag7-Hsp70 complex and decreased the ATP concentration required to dissociate Tag7 from the peptide-binding site of Hsp70. Moreover, HspBP1 inhibited the cytotoxic activity of the Tag7-Hsp70 complex secreted by lymphocytes. HspBP1 was detected in cytotoxic CD8+ lymphocytes. This protein was released simultaneously with Tag7-Hsp70 during interaction of these lymphocytes with tumor cells. The simultaneous secretion of the cytotoxic complex with its inhibitor could be a mechanism protecting normal cells from the cytotoxic effect of this complex.

Peripheral sensory neurons survive in the absence of α- and γ-synucleins

Physiological functions of α-synuclein, a protein implicated in certain types of neurodegeneration, and two other members of the same family, β-synuclein and γ-synuclein, are not clearly understood. It has been suggested that synucleins are involved in intracellular processes associated with survival of neurons and their response to stress, and that changes of synuclein ratio might have deteriorating effects on neurons. In wild-type mice, sensory neurons of the peripheral nervous system express α-synuclein and notably high levels of γ-synuclein, but targeted inactivation of either of these genes has no effect on these neurons. Here we produced double, α-synuclein/γ-synuclein null mutant mice, which develop normally, are fertile, and show no obvious signs of pathology in adulthood. Survival of α/γ-synuclein-deficient peripheral sensory neurons in vivo and in primary tissue culture is indistinguishable from survival of wild-type neurons. The absence of two synucleins does not lead to expression in sensory neurons of the third member of the family, β-synuclein. Therefore, our results demonstrate that neurons with normally high levels of synuclein(s) can develop and survive normally in the absence of any of these proteins. This suggests that other intraneuronal mechanisms and pathways effectively compensate the loss of synuclein function in null mutant animals.

Resistance to tumor necrosis factor induced apoptosis in vitro correlates with high metastatic capacity of cells in vivo.

TNF is one of the cytokines secreted by the cells of the immune system. Our data demonstrate that those cell lines lacking capability to form metastatic tumors in vivo are susceptible to TNF induced apoptosis in vitro. However, cell lines with high metastatic potential are resistant to TNF in vitro. Furthermore, the same cell lines were resistant to cytolytic action of other cytotoxic proteins secreted by LAK cells. Our data showed that TNF resistance in vitro correlates with the increased level of transcription factor NF-kappaB. This finding may provide a tool to improve current protocols of immunotherapy and insights to how tumor cells are or are not killed by LAK cells.

Separation of the pore-forming and cytotoxic activities from natural killer cell cytotoxic factor

The influence of Ca2+ on the cytotoxic activity of natural killer cell cytotoxic factor (NKCF) was analyzed. When the natural killer susceptible cell line K562 was exposed to NKCF in the presence of 5 mM Ca2+, two peaks of cell damage were found. The first peak was observed after 30–40 min of incubation as a result of pore formation on the surface of target cells. The second was a peak of cytolytic activity which appeared after 24 h of incubation. Upon dilution of the NKCF preparation, only the first peak was observed. Therefore, NKCF produced by large granular lymphocytes in response to K562 consists of different proteins and represents pore‐forming and cytolytic activities.